Generating capture probes for spatial analysis

ABSTRACT

The present disclosure relates to compositions and methods for generating capture probes on a substrate for identifying the location of analytes in a biological sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Pat. ApplicationNo. 17/212,681, filed Mar. 25, 2021, which is is a continuationapplication of International Application PCT/US2019/065013, with aninternational filing date of Dec. 6, 2019, which claims priority to U.S.Provisional Pat. Application No. 62/788,871, filed Jan. 6, 2019, U.S.Provisional Pat. Application No. 62/788,885, filed Jan. 6, 2019, U.S.Provisional Pat. Application No. 62/788,897, filed Jan. 6, 2019, U.S.Provisional Pat. Application No. 62/788,906, filed Jan. 6, 2019, U.S.Provisional Pat. Application No. 62/811,495, filed Feb. 27, 2019, U.S.Provisional Pat. Application No. 62/812,219, filed Feb. 28, 2019, U.S.Provisional Pat. Application No. 62/819,449, filed Mar. 15, 2019, U.S.Provisional Pat. Application No. 62/819,456, filed Mar. 15, 2019, U.S.Provisional Pat. Application No. 62/819,470, filed Mar. 15, 2019, U.S.Provisional Pat. Application No. 62/819,478, filed Mar. 15, 2019, U.S.Provisional Pat. Application No. 62/819,495, filed Mar. 15, 2019, U.S.Provisional Pat. Application No. 62/822,554, filed Mar. 22, 2019, U.S.Provisional Pat. Application No. 62/822,565, filed Mar. 22, 2019, U.S.Provisional Pat. Application No. 62/822,566, filed Mar. 22, 2019, U.S.Provisional Pat. Application No. 62/822,592, filed Mar. 22, 2019, U.S.Provisional Pat. Application No. 62/822,605, filed Mar. 22, 2019, U.S.Provisional Pat. Application No. 62/822,606, filed Mar. 22, 2019, U.S.Provisional Pat. Application No. 62/822,610, filed Mar. 22, 2019, U.S.Provisional Pat. Application No. 62/822,627, filed Mar. 22, 2019, U.S.Provisional Pat. Application No. 62/822,680, filed Mar. 22, 2019, U.S.Provisional Pat. Application No. 62/822,722, filed Mar. 22, 2019, U.S.Provisional Pat. Application No. 62/839,212, filed Apr. 26, 2019, U.S.Provisional Pat. Application No. 62/839,223, filed Apr. 26, 2019, U.S.Provisional Pat. Application No. 62/839,264, filed Apr. 26, 2019, U.S.Provisional Pat. Application No. 62/839,320, filed Apr. 26, 2019, U.S.Provisional Pat. Application No. 62/839,346, filed Apr. 26, 2019, U.S.Provisional Pat. Application No. 62/839,575, filed Apr. 26, 2019, U.S.Provisional Pat. Application No. 62/842,463, filed May 2, 2019, U.S.Provisional Pat. Application No. 62/860,993, filed Jun. 13, 2019, U.S.Provisional Pat. Application No. 62/931,779, filed Nov. 6, 2019, U.S.Provisional Pat. Application No. 62/931,587, filed Nov. 6, 2019, U.S.Provisional Pat. Application No. 62/934,883, filed Nov. 13, 2019, U.S.Provisional Pat. Application No. 62/935,043, filed Nov. 13, 2019, U.S.Provisional Pat. Application No. 62/939,488, filed Nov. 22, 2019, andU.S. Provisional Pat. Application No. 62/941,581, filed Nov. 27, 2019.The contents of each of these applications are incorporated herein byreference in their entireties.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submittedelectronically as an XML file named 47706-0007004_SL_ST26.xml. The XMLfile, created on Mar. 16, 2023, is 15,573 bytes in size. The material inthe XML file is hereby incorporated by reference in its entirety.

BACKGROUND

Cells within a tissue of a subject have differences in cell morphologyand/or function due to varied analyte levels (e.g., gene and/or proteinexpression) within the different cells. The specific position of a cellwithin a tissue (e.g., the cell’s position relative to neighboring cellsor the cell’s position relative to the tissue microenvironment) canaffect, e.g., the cell’s morphology, differentiation, fate, viability,proliferation, behavior, and signaling and cross-talk with other cellsin the tissue.

Spatial heterogeneity has been previously studied using techniques thatonly provide data for a small handful of analytes in the contact of anintact tissue or a portion of a tissue, or provide a lot of analyte datafor single cells, but fail to provide information regarding the positionof the single cell in a parent biological sample (e.g., tissue sample).

Arrays that capture spatial information of analytes within a biologicalsample can be prepared in various ways. Arrays can be designed withpluralities of capture probes. Capture probes can be designed to capturea substantial population of analytes (e.g., mRNA) or to capture specific(e.g., gene specific) analytes. The methods described herein provide forways of preparing arrays with capture probes and capturing analytes.

SUMMARY

Provided herein are methods of generating a spatial array including: (a)providing an array including a plurality of oligonucleotides, where the3′ end of an oligonucleotide of the plurality of oligonucleotides isattached to a substrate, (b) providing a plurality of primers, where aprimer of the plurality of primers is substantially complementary to aportion of the oligonucleotide, (c) extending the primer using theoligonucleotide as a template, thereby generating a firstoligonucleotide with a free 3′ end, (d) extending the firstoligonucleotide to produce a 3′ overhang, (e) providing a splintoligonucleotide that hybridizes to the 3′ end of the firstoligonucleotide, and (f) ligating a second oligonucleotide to the 3′ endof the first oligonucleotide, where the second oligonucleotide includesa sequence that is substantially complementary to a portion of thesplint oligonucleotide, and where the second oligonucleotide includes acapture domain, thereby generating a spatial array.

In some embodiments of methods of generating a spatial array, theoligonucleotide of the plurality of oligonucleotides includes a spatialbarcode. In some embodiments, the oligonucleotide of the plurality ofoligonucleotides includes a first constant sequence. In some embodimentsof methods of generating a spatial array, the oligonucleotide of theplurality of oligonucleotides further includes a second constantsequence. In some embodiments of methods of generating a spatial array,the oligonucleotide of the plurality of oligonucleotides has a length ofbetween 20 and 60 nucleotides.

In some embodiments of methods of generating a spatial array, the secondoligonucleotide further includes a constant sequence, and where theconstant sequence is substantially complementary to a portion of thesplint oligonucleotide. In some embodiments of methods of generating aspatial array, the second oligonucleotide further includes a uniquemolecular identifier. In some embodiments of methods of generating aspatial array, the second oligonucleotide further includes a spatialbarcode.

In some embodiments of methods of generating a spatial array, thecapture domain includes a poly-T sequence. In some embodiments ofmethods of generating a spatial array, the capture domain includes agene-specific sequence.

In some embodiments of methods of generating a spatial array, the 3′ endoverhang of step (d) is produced by an enzyme. In some embodiments ofmethods of generating a spatial array, the enzyme is atemplate-independent polymerase. In some embodiments of methods ofgenerating a spatial array, the template-independent polymerase isterminal deoxynucleotidyl transferase (TdT) or poly(A) polymerase.

In another aspect, provided herein are methods for determining thelocation of an analyte in a biological sample including (a) contactingthe biological sample with a spatial array produced by any of thevariety of methods herein (b) permeabilizing the biological sample toallow the analyte in the biological sample to specifically bind with acapture domain on the spatial array, and (c) identifying the analytespecifically bound to the capture domain, thereby determining thespatial location of the analyte in the biological sample.

In some embodiments of methods for determining the location of ananalyte in a biological sample, a second analyte in the biologicalsample interacts with a second capture domain on the spatial array, andstep (c) includes identifying the second analyte specifically bound tothe second capture domain, thereby determining the spatial location ofthe analyte in the second biological sample.

In another aspect, provided herein are methods for preparing a spatialarray, including (a) contacting an array including one or morepluralities of first oligonucleotides with a hydrogel including one ormore pluralities of second oligonucleotides, where a firstoligonucleotide of the one or more pluralities of first oligonucleotidesincludes a capture domain, a second oligonucleotide of the one or morepluralities of second oligonucleotides includes a capture domain, orboth, (b) attaching the first oligonucleotide to the secondoligonucleotide to generate a capture probe including a capture domain,(c) reducing the size of the hydrogel including the capture probe byremoving water to form a shrunken hydrogel including the capture probe,thereby generating the spatial array.

In some embodiments of methods for preparing a spatial array, the one ormore pluralities of second oligonucleotides are immobilized in thehydrogel. In some embodiments of methods for preparing a spatial array,the immobilized second oligonucleotides are partitioned such that afirst population of the immobilized second oligonucleotides ispartitioned away from another population of immobilized secondoligonucleotides.

In some embodiments of methods for preparing a spatial array, theimmobilized second oligonucleotides are partitioned into a plurality ofsecond oligonucleotide partitions such that different populations of theimmobilized second oligonucleotides are partitioned away from eachother. In some embodiments of methods for preparing a spatial array, afirst immobilized second oligonucleotide of a partitioned population ofimmobilized second oligonucleotides is different from a secondimmobilized second oligonucleotide of another partitioned population ofimmobilized second oligonucleotides. In some embodiments of methods forpreparing a spatial array, the first immobilized second oligonucleotide,the second immobilized second oligonucleotide, or both include a spatialbarcode.

In some embodiments of methods for preparing a spatial array, the firstimmobilized second oligonucleotide of a partitioned population ofimmobilized second oligonucleotides includes a spatial barcode that isdifferent from a spatial barcode of the second immobilized secondoligonucleotide of another partitioned population of immobilized secondoligonucleotides.

In some embodiments of methods for preparing a spatial array, one ormore second oligonucleotide partitions include a population ofimmobilized capture probes including a spatial barcode different from aspatial barcode of the population of immobilized capture probes ofanother second oligonucleotide partition.

In some embodiments of methods for preparing a spatial array, the one ormore pluralities of first oligonucleotides are partitioned. In someembodiments of methods for preparing a spatial array, the one or morepluralities of first oligonucleotides are partitioned into a firstoligonucleotide partitions. In some embodiments of methods for preparinga spatial array, one or more first oligonucleotide partitions include apopulation of first oligonucleotides that is different from firstoligonucleotides in another first oligonucleotide partition. In someembodiments of methods for preparing a spatial array, a firstoligonucleotide of a population of first oligonucleotides in one or morefirst oligonucleotide partitions includes a spatial barcode. In someembodiments of methods for preparing a spatial array, the firstoligonucleotide of a population of first oligonucleotides in one or morefirst oligonucleotide partitions includes a spatial barcode that isdifferent from the spatial barcode of a first oligonucleotide of apopulation of first oligonucleotides in another first oligonucleotidepartition. In some embodiments of methods for preparing a spatial array,a spatial barcode of the first oligonucleotide of a population of firstoligonucleotides in one or more first oligonucleotide partitions isdifferent from a spatial barcode of a second oligonucleotide of apopulation of second oligonucleotides in one or more secondoligonucleotide partitions. In some embodiments of methods for preparinga spatial array, a first oligonucleotide partition includes a firstpopulation of first oligonucleotides including a spatial barcode that isdifferent from a spatial barcode of a second population of firstoligonucleotides in another first oligonucleotide partition.

In some embodiments of methods for preparing a spatial array, thereduction in the size of the hydrogel in step (c) is isometric. In someembodiments of methods for preparing a spatial array, the removal ofwater in step (c) includes a dehydrating solvent. In some embodiments ofmethods for preparing a spatial array, the solvent includes at least oneof an alcohol, a ketone, an aldehyde, acetonitrile, or a combinationthereof. In some embodiments of methods for preparing a spatial array,the solvent includes at least one of glycerol, ethanol, acetonitrile,acetone, 2-propanol, and a combination thereof. In some embodiments, theremoval of water in step (c) includes the use of a salt. In someembodiments of methods for preparing a spatial array, the salt includesan inorganic salt. In some embodiments of methods for preparing aspatial array, the removal of water in step (c) includes an acid. Insome embodiments of methods for preparing a spatial array, the removalof water in step (c) includes a dehydrating process.

In some embodiments of methods for preparing a spatial array, theshrunken hydrogel is about 2 to about 10 fold smaller in a lineardimension than the size of the hydrogel prior to step (c) pre-shrunkhydrogel. In some embodiments of methods for preparing a spatial array,the shrunken hydrogel is at least 2-fold smaller in a linear dimensionthan the pre-shrunk hydrogel. In some embodiments of methods forpreparing a spatial array, the shrunken hydrogel is at least 4-foldsmaller in a linear dimension than the pre-shrunk hydrogel. In someembodiments of methods for preparing a spatial array, the shrunkenhydrogel is at least 8-fold smaller in a linear dimension than thepre-shrunk hydrogel.

In some embodiments of methods for preparing a spatial array, attachingincludes a polymerase chain reaction. In some embodiments of methods forpreparing a spatial array, the hydrogel includes a DNA polymerase, aplurality of primers, and a plurality of nucleotides. In someembodiments of methods for preparing a spatial array, members of aplurality of first oligonucleotides are cross-linked to the hydrogel. Insome embodiments of methods for preparing a spatial array, the captureprobe is amplified. In some embodiments of methods for preparing aspatial array, the capture probe is amplified prior to the reduction ofthe size of the hydrogel.

In some embodiments of methods for preparing a spatial array, thehydrogel is prepared from hydrogel subunits including at least one of:acrylamide, bis-acrylamide, a poly(ethylene glycol), a polyaliphaticpolyurethane, a polyether polyurethane, a polyester polyurethane, apolyethylene copolymer, a polyamide, a polyvinyl alcohol, polypropyleneglycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide,poly(hydroxyethyl acrylate), poly(hydroxyethyl methacrylate), collagen,hyaluronic acid, chitosan, dextran, agarose, gelatin, alginate, aprotein polymer, methylcellulose, and a combination thereof.

In some embodiments of methods for preparing a spatial array, the firstoligonucleotide of the one or more pluralities of first oligonucleotidesincludes a spatial barcode. In some embodiments of methods for preparinga spatial array, the first oligonucleotide of the one or morepluralities of first oligonucleotides includes a unique molecularidentifier. In some embodiments of methods for preparing a spatialarray, the first oligonucleotide of the one or more pluralities of firstoligonucleotides includes a spatial barcode and a capture domain.

In another aspect, provided herein are methods for determining thelocation of an analyte in a biological sample, including (a) providing aspatial array of any of the variety of methods described herein, (b)contacting a biological sample with a spatial array of any one of thespatial arrays described herein under conditions that allow the analyteto specifically bind to a capture probe of the spatial array, and (c)determining the identity of the analyte specifically bound to thecapture probe, thereby determining the location of the analyte in thebiological sample.

In some embodiments of methods for determining the location of ananalyte in a biological sample, the biological sample is a tissuesample. In some embodiments of methods for determining the location ofan analyte in a biological sample, the tissue sample is a fresh-frozentissue sample. In some embodiments of methods for determining thelocation of an analyte in a biological sample, the tissue sampleincludes a tumor cell. In some embodiments of methods for determiningthe location of an analyte in a biological sample, the tissue sampleincludes a tissue section.

In some embodiments, methods for determining the location of an analytein a biological sample further includes migrating the analyte in thebiological sample to the capture probe. In some embodiments of methodsfor determining the location of an analyte in a biological sample,migrating includes passive migration. In some embodiments of methods fordetermining the location of an analyte in a biological sample, migratingincludes active migration.

In some embodiments of methods for determining the location of ananalyte in a biological sample, the analyte includes at least one ofRNA, DNA, protein, lipid, peptide, metabolite, small molecule, and aanalyte capture agent.

In another aspect, provided herein are methods of generating an arrayincluding (a) providing a substrate including a plurality of featuresimmobilized on the substrate, where a feature of the plurality offeatures includes a first oligonucleotide, and where the firstoligonucleotide includes a first spatial barcode, (b) delivering asecond oligonucleotide to the first oligonucleotide, where the secondoligonucleotide includes a second spatial barcode and a capture domain,and (c) attaching the first oligonucleotide and the secondoligonucleotide, thereby generating a capture probe including the firstspatial barcode, the second spatial barcode, and the capture domain.

In some embodiments of methods of generating an array, the firstoligonucleotide is associated with one of a number of different firstspatial barcodes. In some embodiments of methods of generating an array,the second oligonucleotide is associated with one of a number ofdifferent second spatial barcodes.

In some embodiments of methods of generating an array, step (c) includesligating the first oligonucleotide and the second oligonucleotide. Insome embodiments of methods of generating an array, the ligatingincludes the use of a ligase. In some embodiments of methods ofgenerating an array, the first oligonucleotide and the secondoligonucleotide are attached to one another by a chemical couplingreaction.

In some embodiments of methods of generating an array, the firstoligonucleotide includes a 3′ end that is masked by a protective group.In some embodiments of methods of generating an array, the protectivegroup is removed prior to delivering the second oligonucleotide to thefirst oligonucleotide. In some embodiments of methods of generating anarray, the protective group is a photocleavable protective group. Insome embodiments of methods of generating an array, the photocleavableprotective group is removed by exposing the first oligonucleotide tolight. In some embodiments of methods of generating an array, thephotocleavable protective group coupled to the first oligonucleotide isprotected by applying a photomask.

In some embodiments of methods of generating an array, a first featureis separated from other features of the plurality by one or morephysical barriers, where the one or more physical barriers form one ormore chambers, and where the first oligonucleotide is delivered to achamber of the one or more chambers.

In some embodiments of methods of generating an array, the one or morephysical barriers include a hydrophobic surface. In some embodiments ofmethods of generating an array, the one or more physical barriersinclude one or more chamber walls having a tapered end in contact withthe substrate. In some embodiments of methods of generating an array,the first oligonucleotide includes a unique molecular identifier.

In another aspect, provided herein are methods of generating an arrayincluding (a) providing a substrate including two or more partitions,where a first partition of the two or more partitions includes a featureimmobilized on the substrate, where the feature includes a firstoligonucleotide, and where the first oligonucleotide includes a firstspatial barcode, (b) delivering a second oligonucleotide to the firstpartition, where the second oligonucleotide includes a second spatialbarcode and a capture domain, and (c) attaching the firstoligonucleotide and the second oligonucleotide in the first partition,thereby generating a capture probe including the first spatial barcode,the second spatial barcode, and the capture domain in the firstpartition.

In some embodiments, methods of generating an array further include (d)delivering a third oligonucleotide to a second partition of the two ormore partitions, where the third oligonucleotide includes a thirdspatial barcode and a second capture domain, where the second partitionincludes a second feature, where the second feature includes a fourtholigonucleotide including the first spatial barcode, and (e) attachingthe third oligonucleotide and the fourth oligonucleotide, therebygenerating a second capture probe including the first spatial barcode,the third spatial barcode, and the second capture domain in the secondpartition.

In some embodiments of methods of generating an array, the two or morepartitions are separated by one or more physical barriers. In someembodiments of methods of generating an array, the one or more physicalbarriers are printed on the substrate. In some embodiments of methods ofgenerating an array, the one or more physical barriers form one or morechambers including one or more chamber walls. In some embodiments ofmethods of generating an array, the one or more chamber walls include ahydrophobic surface. In some embodiments, the one or more chamber wallsinclude a tapered end in contact with the substrate. In some embodimentsof methods of generating an array, the second oligonucleotide isdelivered using a microfluidic device.

In another aspect, provided herein are methods for enriching apopulation of capture probes including (a) providing a plurality ofcapture probes where: (i) members of the plurality of capture probesinclude a plurality of different spatial barcodes, and (ii) a targetcapture probe of the population of capture probes to be enrichedincludes a target spatial barcode, and (b) enriching a population oftarget capture probes including the target spatial barcode from theplurality of capture probes.

In another aspect, provided herein are methods for determining thelocation of an analyte in a biological sample including (a) providing asubstrate including a plurality of capture probes, where a subset of theplurality of capture probes is attached at a location on the substrate,and where two or more capture probes of the subset of capture probesincludes a first common spatial barcode, (b) contacting the biologicalsample with the substrate such that a capture probe of the subset ofcapture probes specifically binds to the analyte from the biologicalsample, where the analyte that is specifically bound to the captureprobe includes the first common spatial barcode or a complement thereof,(c) identifying a region of interest within the biological sample, wherethe region of interest includes the analyte specifically bound to thecapture probe, and (d) enriching the analyte specifically bound to thecapture probe to determine the location of the analyte in the biologicalsample.

In some embodiments of methods for determining the location of ananalyte in a biological sample, the substrate further includes a secondsubset of capture probes attached at a second location on the substrate,where two or more capture probes of the second subset of capture probesinclude a second common spatial barcode that is different from the firstcommon spatial barcode.

In some embodiments of methods for determining the location of ananalyte in a biological sample, the substrate further includes a thirdsubset of capture probes attached at a third position on the substrate,where two or more capture probes of the third subset of capture probesincludes a third common spatial barcode that is different from the firstcommon spatial barcode and the second common spatial barcode. In someembodiments of methods for determining the location of an analyte in abiological sample, the capture probe includes a capture domain.

In some embodiments of methods for determining the location of ananalyte in a biological sample, the analyte includes at least one ofRNA, DNA, and a analyte capture agent. In some embodiments of methodsfor determining the location of an analyte in a biological sample, theanalyte capture agent includes an antigen binding molecule. In someembodiments of methods for determining the location of an analyte in abiological sample, the analyte capture agent includes a regionconfigured to couple or specifically bind to the capture domain of thecapture probe.

In some embodiments of methods for determining the location of ananalyte in a biological sample, the region of interest is identified byperforming shallow sequencing of at least a subset of a plurality ofanalytes specifically bound to capture probes, and identifying the firstcommon spatial barcode associated with the region of interest. In someembodiments of methods for determining the location of an analyte in abiological sample, the region of interest is identified by at least oneof immunostaining, immunohistochemistry, microscopy, visual inspection,and combinations thereof.

In some embodiments of methods for determining the location of ananalyte in a biological sample, step (d) includes performing a nucleicacid extension reaction using (i) the analyte specifically bound to thecapture probe and (ii) a primer including a sequence specific for thefirst common spatial barcode or a complement thereof to generate anenriched plurality of the analyte specifically bound to the captureprobe(s) including the first common barcode or a complement thereof.

In some embodiments of methods for determining the location of ananalyte in a biological sample, the analyte is amplified by performing apolymerase chain reaction (PCR).

In some embodiments, methods for determining the location of an analytein a biological sample further include enriching a population of captureprobes present in a subregion of interest (a) where the subregion ofinterest is located within the region of interest, where the subregionof interest includes a sub-plurality of capture probes, and where: (i)two or more capture probes of the sub-plurality of capture probesinclude a first common sub-spatial barcode, and (ii) the first commonsub-spatial barcode sequence is associated with the subregion ofinterest, and (b) enriching a population of capture probe including thefirst common sub-spatial barcode.

In some embodiments, methods for determining the location of an analytein a biological sample further include enriching a population of captureprobes present in a sub-subregion of interest (a) where thesub-subregion of interest is located within the subregion of interest,where the sub-subregion of interest includes a sub-sub-plurality ofcapture probes, and where: (i) two or more capture probes of the sub-subplurality of capture probes include a first common sub-sub spatialbarcode, and (ii) the first common sub-sub spatial barcode is associatedwith the sub-sub region of interest, and (b) enriching the population ofcapture probes including the first sub-sub spatial barcode.

In some embodiments of methods for determining the location of ananalyte in a biological sample, the first common spatial barcode isassociated with a single cell of the biological sample.

In some embodiments of methods for determining the location of ananalyte in a biological sample, the analyte includes an mRNA. In someembodiments of methods for determining the location of an analyte in abiological sample, the analyte includes a protein. In some embodimentsof methods for determining the location of an analyte in a biologicalsample, the analyte includes an antibody. In some embodiments of methodsfor determining the location of an analyte in a biological sample, theanalyte includes DNA. In some embodiments of methods for determining thelocation of an analyte in a biological sample, the DNA encodes a T-cellreceptor. In some embodiments of methods for determining the location ofan analyte in a biological sample, the DNA includes one or more regionsof chromatin in the biological sample.

In another aspect, provided herein are methods for generating a spatialarray including (a) providing a substrate including a plurality of firstoligonucleotides, where a first oligonucleotide of the plurality offirst oligonucleotides is attached to a region of the substrate throughits 3′-end or its 5′-end, (b) attaching a second oligonucleotide to anunattached end of the first oligonucleotide, where the secondoligonucleotide includes a spatial barcode.

In some embodiments, methods for generating a spatial array furtherinclude after (b): (c) attaching a third oligonucleotide segment to anunattached end of the second oligonucleotide.

In some embodiments of methods for generating a spatial array, the firstoligonucleotide is attached to the substrate through its 5′-end and thesecond oligonucleotide is generated using spatially-directed in situsynthesis on the 3′-end of the first oligonucleotide. In someembodiments of methods for generating a spatial array, the firstoligonucleotide is attached to the substrate through its 3′-end and thesecond oligonucleotide is generated using spatially-directed in situsynthesis on the 5′-end of the first oligonucleotide.

In some embodiments of methods for generating a spatial array,phosphodiester and exocyclic amine moieties of the first oligonucleotideare protected.

In some embodiments of methods for generating a spatial array, thesecond oligonucleotide has a length selected from 8 nucleotides, 9nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29nucleotides, or 30 nucleotides.

In some embodiments of methods for generating a spatial array, the insitu synthesis is light-directed synthesis.

In some embodiments of methods for generating a spatial array, theregion of the substrate is less than about 2500 µm², less than about1600 µm², less than about 400 µm², less than about 100 µm², less thanabout 50 µm², less than about 25 µm², less than about 15 µm², less thanabout 10 µm², or less than about 5 µm².

In some embodiments of methods for generating a spatial array, two ormore second oligonucleotides are attached to two or more firstoligonucleotides, where a member of the two or more secondoligonucleotides differs from another member of the two or more secondoligonucleotides.

In some embodiments of methods for generating a spatial array, the firstoligonucleotide includes a protected phosphodiester and exocyclic aminemoieties.

In some embodiments of methods for generating a spatial array, thesubstrate further includes a substantially complementary oligonucleotidehybridized to the first oligonucleotide.

In some embodiments of methods for generating a spatial array, the firstoligonucleotide has a length of at least about 2 nucleotides, 5nucleotides, 8 nucleotides, 10 nucleotides, 15 nucleotides, 18nucleotides, 20 nucleotides, 22 nucleotides, 25 nucleotides, 27nucleotides, 30 nucleotides, 33 nucleotides, or 35 nucleotides. In someembodiments of methods for generating a spatial array, the firstoligonucleotide has a length of about 2 to 45 nucleotides, 5 to 40nucleotides, 15 to 40 nucleotides, 25 to 40 nucleotides, 25 to 35nucleotides, or 27 to 33 nucleotides.

In some embodiments of methods for generating a spatial array, the firstoligonucleotide is prepared by synthesis off of the substrate and thefirst oligonucleotide is subsequently attached to the substrate.

In some embodiments of methods for generating a spatial array, thephosphodiester and exocyclic amine moieties of the first oligonucleotideare protected prior to attachment of the first oligonucleotide to thesubstrate. In some embodiments of methods for generating a spatialarray, the phosphodiester and exocyclic amine moieties of the firstoligonucleotide are protected after attachment of the firstoligonucleotide to the substrate.

In some embodiments of methods for generating a spatial array, the firstoligonucleotide is prepared by in situ synthesis on the substrate. Insome embodiments of methods for generating a spatial array, the in situsynthesis is not light-directed synthesis. In some embodiments ofmethods for generating a spatial array, the in situ synthesis is notspatially-directed synthesis.

In some embodiments of methods for generating a spatial array, the firstoligonucleotide includes a sequence including a cleavage site. In someembodiments of methods for generating a spatial array, the firstoligonucleotide is attached to the substrate through a linker. In someembodiments of methods for generating a spatial array, a chemical groupincluding: silane, amine, peptide, polyethylene glycol, biotin,desthiobiotin, streptavidin, thiol, acrydite, azide, digoxigenin,hexynyl, or a combination thereof. In some embodiments of methods forgenerating a spatial array, the linker is a branched linker or adendrimeric linker.

In some embodiments of methods for generating a spatial array, the thirdoligonucleotide is attached to the second oligonucleotide by ligation.

In some embodiments of methods for generating a spatial array, anyprotected moieties of the first and second oligonucleotides aredeprotected prior to ligating the third oligonucleotide to the secondoligonucleotide.

In some embodiments of methods for generating a spatial array, theplurality of third oligonucleotides have a length of at least about 5nucleotides, 7 nucleotides, 9 nucleotides, 13 nucleotides, 20nucleotides, 25 nucleotides, 27 nucleotides, 30 nucleotides, 33nucleotides, or 35 nucleotides. In some embodiments of methods forgenerating a spatial array, the plurality of third oligonucleotides havea length of about 5 to 40 nucleotides, 5 to 20 nucleotides, 5 to 10nucleotides, 10 to 35 nucleotides, 15 to 30 nucleotides, 25 to 35nucleotides, or 27 to 33 nucleotides.

In some embodiments of methods for generating a spatial array, the thirdoligonucleotide includes a capture domain of at least 6 nucleotides inlength at the 3′-end. In some embodiments of methods for generating aspatial array, the capture domain includes a poly-T sequence. In someembodiments of methods for generating a spatial array, the thirdoligonucleotide includes a random sequence of 6 to 20 nucleotides inlength.

In another aspect, provided herein are compositions including aplurality of oligonucleotides attached to a plurality of regions on asubstrate, where an oligonucleotide of the plurality of oligonucleotidesincludes: a first oligonucleotide of 2 to 50 nucleotides in lengthattached to the substrate through its 3′-end or its 5′-end, and a secondoligonucleotide of 8 to 30 nucleotides in length that is attached to anunattached end of the first oligonucleotide, where the secondoligonucleotide includes a spatial barcode.

In another aspect, provided herein are compositions where the firstoligonucleotide and the second oligonucleotide include protectedphosphodiester and exocyclic amine moieties.

In another aspect, provided herein are compositions where the firstoligonucleotide is attached to the substrate through its 5′-end and thesecond oligonucleotide is attached to the 3′-end of the firstoligonucleotide.

In another aspect, provided herein are compositions where the firstoligonucleotide is attached to the substrate through its 3′-end and thesecond oligonucleotide is attached to the 5′-end of the firstoligonucleotide.

In some embodiments, compositions where the first oligonucleotide isattached to the substrate through its 3′-end and the secondoligonucleotide is attached to the 5′-end of the first oligonucleotidefurther include a third oligonucleotide of 5 to 40 nucleotides in lengththat is attached to an unattached end of the second oligonucleotide,where the third oligonucleotide includes a capture domain.

In some embodiments, compositions where the first oligonucleotide isattached to the substrate through its 3′-end and the secondoligonucleotide is attached to the 5′-end of the first oligonucleotide,the second oligonucleotide has a length selected from 8 nucleotides, 9nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29nucleotides, or 30 nucleotides.

In some embodiments, compositions where the first oligonucleotide isattached to the substrate through its 3′-end and the secondoligonucleotide is attached to the 5′-end of the first oligonucleotide,the regions are less than about 2500 µm², less than about 1600 µm², lessthan about 400 µm², less than about 100 µm², less than about 50 µm²,less than about 25 µm², less than about 15 µm², less than about 10 µm²,less than about 5 µm², or less than about 1 µm².

In some embodiments, compositions where the first oligonucleotide isattached to the substrate through its 3′-end and the secondoligonucleotide is attached to the 5′-end of the first oligonucleotide,the length of the oligonucleotide in each of the regions is at least 15nucleotides, at least 20 nucleotides, at least 40 nucleotides, at least50 nucleotides, or at least 60 nucleotides.

In some embodiments, compositions where the first oligonucleotide isattached to the substrate through its 3′-end and the secondoligonucleotide is attached to the 5′-end of the first oligonucleotide,each of the regions is from 15 to 100 nucleotides, 20 to 100nucleotides, 40 to 100 nucleotides, from 50 to 90 nucleotides, or from60 to 80 nucleotides.

In another aspect, provided herein are methods of preparing a spatialarray including (a) providing a substrate, (b) immobilizing a pluralityof sets of primers on the substratet, where a first set of the pluralityof sets of primers includes a first primer and a second primer, (c)providing a template oligonucleotide including a reverse complement of aspatial barcode domain, a reverse complement of a capture domain, afirst hybridization domain capable of hybridizing to the first primer,and a second hybridization domain, where a reverse complement of thesecond hybridization domain is capable of hybridizing to the secondprimer, (d) hybridizing the template oligonucleotide to the firstprimer, (e) extending the first primer using the templateoligonucleotide as a template to generate a capture probe inclduing thefirst primer, the spatial barcode, and the capture domain, which captureprobe is immobilized on the substrate, and (f) releasing the templateoligonucleotide from the capture probe, thereby producing the spatialarray.

In some embodiments, methods of preparing a spatial array furtherinclude amplifying the capture probe to create a cluster of one or morecapture probes.

In some embodiments of methods of preparing a spatial array, amplifyingincludes (a) hybridizing the capture probe to the second primer, (b)extending the second primer using the capture probe as a template togenerate a copy of the template oligonucleotide that is immobilized onthe substrate, (c) hybridizing the copy of the template oligonucleotideto a first primer of a second set of the plurality of primers, and (d)extending the first primer of the second set of the plurality of primersusing the copy of template oligonucleotide as a template to generate acopy of the capture probe that is immobilized on the substrate, wherethe cluster of capture probes includes the capture probe and the copy ofthe capture probe.

In some embodiments of methods of preparing a spatial array, thetemplate oligonucleotide includes a reverse complement of a uniquemolecular identifier (UMI), and where the generated capture probeincludes the UMI.

In some embodiments of methods of preparing a spatial array, the firstprimer is affixed to the substrate at a 5′ end of the first primer. Insome embodiments of methods of preparing a spatial array, the firstprimer includes a sequence that is complementary to the templateoligonucleotide. In some embodiments of methods of preparing a spatialarray, the first primer includes a poly (A) domain. In some embodimentsof methods of preparing a spatial array, the first primer includescleavage domain.

In some embodiments of methods of preparing a spatial array, the secondprimer is affixed to the substrate at a 5′ end of the second primer. Insome embodiments of methods of preparing a spatial array, the secondprimer includes a cleavage domain.

In some embodiments of methods of preparing a spatial array, the copy ofthe template oligonucleotide is released by cleaving the cleavage domainafter the copy of the capture probe is generated. In some embodiments ofmethods of preparing a spatial array, the template oligonucleotide isattached to a bead.

In some embodiments of methods of preparing a spatial array, detachingthe bead from the template oligonucleotide after the step of extendingthe first primer using the template oligonucleotide as a template togenerate a capture probe including the first primer, the spatialbarcode, and the capture domain, which capture probe is immobilized onthe substrate.

In some embodiments of methods of preparing a spatial array, the bead isa gel bead. In some embodiments of methods of preparing a spatial array,the bead is 2 microns or less in diameter. In some embodiments ofmethods of preparing a spatial array, the bead is 1 micron or less indiameter. In some embodiments of methods of preparing a spatial array,the bead is removed prior to the step of extending the first primer. Insome embodiments of methods of preparing a spatial array, the bead isremoved after the step of extending the first primer.

In some embodiments of methods of preparing a spatial array, all or aportion of the sequence of the generated capture probe is determined. Insome embodiments of methods of preparing a spatial array, all or aportion of the sequence of the generated capture probe is determinedafter the template oligonucleotide is released.

In some embodiments of methods of preparing a spatial array, all or aportion of the sequence of the generated capture probe is determined insitu. In some embodiments of methods of preparing a spatial array, allor a portion of the sequence of the generated capture probe isdetermined by in situ sequencing. In some embodiments of methods ofpreparing a spatial array, the in situ sequencing is performed viasequencing-by-synthesis (SBS), sequential fluorescence hybridization,sequencing by ligation, nucleic acid hybridization, or high-throughputdigital sequencing techniques.

In some embodiments of methods of preparing a spatial array, the capturedomain includes a sequence complementary to an analyte of interest. Insome embodiments of methods of preparing a spatial array, the capturedomain includes a poly(T) domain.

In some embodiments of methods of preparing a spatial array, thesubstrate includes a glass slide. In some embodiments of methods ofpreparing a spatial array, the glass slide is a treated glass slide.

In another aspect, provided herein are methods for determining alocation of an analyte in a biological sample including (a) preparing aspatial array using any of the variety of methods described herein, (b)decoding the one or more capture probes by determining all or a portionof the sequence of the spatial barcode of the one or more captureprobes, thereby associating the spatial barcode of the one or morecapture probe with a location on the spatial array, (c) contacting thebiological sample to the spatial array under conditions where theanalyte specifically binds to capture probe(s), (d) extending the one ormore capture probes via a polymerization reaction using the analyte as atemplate to generate an extended one or more capture probes includingthe one or more capture probes and the reverse complement of theanalyte, and (e) determining the sequence of the extended one or morecapture probes, and associating the spatial barcode of the one or morecapture probes with the analyte, thereby determining the location of theanalyte in the biological sample.

In some embodiments of methods for determining a location of an analytein a biological sample, one or more capture probes includes a templateoligonucleotide and a bead. In some embodiments, methods for determininga location of an analyte in a biological sample further includedetaching the bead from the template oligonucleotide after the step ofextending the one or more capture probes via a polymerization reactionusing the analyte as a template to generate an extended one or morecapture probes including the one or more capture probes and the reversecomplement of the analyte.

In some embodiments of methods for determining a location of an analytein a biological sample, the first primer and the second primer are at aratio of between 1:1.5 and 1.5:1. In some embodiments of methods fordetermining a location of an analyte in a biological sample, the firstprimer and the second primer are less than 5 µm apart.

In another aspect, provided herein are spatial arrays produced by any ofthe methods described herein.

In another aspect, provided herein are spatial array including (a) asubstrate, and (b) a plurality of sets of primers immobilized on thesubstrate, where a first set of the plurality of sets of primersincludes a first primer and a second primer, and where the first primerincludes a sequence that is complementary to a capture probe.

In some embodiments, the spatial array further includes a templateoligonucleotide including a reverse complement of a unique molecularidentifier (UMI), and where the capture probe includes the UMI.

In some embodiments of spatial arrays provided herein, the first primeris affixed to the substrate at a 5′ end of the first primer. In someembodiments of spatial arrays provided herein, the first primer includesa sequence that is complementary to the template oligonucleotide. Insome embodiments of spatial arrays provided herein, the first primerincludes poly (A) domain. In some embodiments of spatial arrays providedherein, the first primer includes a cleavage domain.

In some embodiments of spatial arrays provided herein, the second primeris affixed to the substrate at a 5′ end of the second primer. In someembodiments of spatial arrays provided herein, the second primerincludes a cleavage domain.

In some embodiments of spatial arrays provided herein, the templateoligonucleotide is attached to a bead. In some embodiments of spatialarrays provided herein, the bead is a gel bead. In some embodiments ofspatial arrays provided herein, the bead is 2 microns or less indiameter. In some embodiments of spatial arrays provided herein, thebead is 1 micron or less in diameter.

In some embodiments of spatial arrays provided herein, the substrateincludes a glass slide. In some embodiments of spatial arrays providedherein, the glass slide is a treated glass slide.

In some embodiments of spatial arrays provided herein, the first primerand the second primer are at a ratio of between 1:1.5 and 1.5:1. In someembodiments of spatial arrays provided herein, the first primer and thesecond primer are less than 5 µm apart.

In another aspect, provided herein are methods of generating a spatialarray including (a) providing a substrate including an arrayed pluralityof acceptor oligonucleotides, where an acceptor oligonucleotide of theplurality of acceptor oligonucleotides includes a spatial barcode and afirst ligation handle, and where the 5′ end of the acceptoroligonucleotide is attached to the substrate, (b) providing a splintoligonucleotide including a sequence complementary to the first ligationhandle and a sequence complementary to a donor oligonucleotide, and (c)ligating a donor oligonucleotide including a capture domain to the 3′end the acceptor oligonucleotide to generate a capture probe, therebygenerating a spatial array.

In some embodiments of methods of generating a spatial array, theacceptor oligonucleotides further includes a cleavage domain, a uniquemolecular identifier, a functional sequence, or any combination thereof.

In some embodiments of methods of generating a spatial array, thesequence complementary to the capture domain of splint oligonucleotideis a poly(A) sequence. In some embodiments, the sequence complementaryto the capture domain of the splint oligonucleotide is a gene-specificsequence. In some embodiments, the gene-specific sequence is aPenk-specific sequence. In some embodiments, the sequence complementaryto the capture domain of the splint oligonucleotide is a randomsequence. In some embodiments, the random sequence is a hexamer.

In some embodiments, methods of generating a spatial array furtherinclude contacting a biological sample to the spatial array after step(c). In some embodiments, methods of generating a spatial array furtherinclude permeabilizing the biological sample to allow the analyte in thebiological sample to interact with the capture probe.

In some embodiments, methods of generating a spatial array furtherinclude migrating the analyte in the biological sample to the pluralityof capture probes. In some embodiments of methods of generating aspatial array, the migrating includes passive migration. In someembodiments of methods of generating a spatial array, the migratingincludes active migration.

In another aspect, provided herein are methods of generating a spatialarray including (a) providing a substrate including an arrayed pluralityof acceptor oligonucleotides, where an acceptor oligonucleotide of theplurality of acceptor oligonucleotides includes a spatial barcode and afirst ligation handle, and where the 5′ end of the acceptoroligonucleotide is attached to the substrate, (b) providing a splintoligonucleotide including a sequence complementary to the first ligationhandle and a sequence complementary to a second ligation handle, and (c)ligating a donor oligonucleotide including the second ligation handleand a capture domain to the 3′ end the acceptor oligonucleotide togenerate a capture probe, thereby generating a spatial array.

In some embodiments of methods of generating a spatial array, theacceptor oligonucleotides further includes a cleavage domain, a uniquemolecular identifier, a functional sequence, or any combination thereof.

In some embodiments of methods of generating a spatial array, thecapture domain includes a poly(T) sequence. In some embodiments ofmethods of generating a spatial array, the capture domain includes agene-specific sequence. In some embodiments of methods of generating aspatial array, the gene-specific sequence is a Penk-specific sequence.In some embodiments of methods of generating a spatial array, thecapture domain includes a random sequence. In some embodiments ofmethods of generating a spatial array, the random sequence is a hexamer.

In some embodiments, methods of generating a spatial array furtherinclude contacting a biological sample to the spatial array after step(c). In some embodiments, methods of generating a spatial array furtherinclude permeabilizing the biological sample to allow an analyte in thebiological sample to interact with the capture probe.

In some embodiments, methods of generating a spatial array furtherinclude migrating the analyte in the biological sample to the captureprobe. In some embodiments of methods of generating a spatial array, themigrating includes passive migration. In some embodiments of methods ofgenerating a spatial array, the migrating includes active migration.

In another aspect, provided herein are methods of generating a captureprobe bound to substrate including dispensing a second solution onto aportion of a surface of a substrate coated with a first solution, whereafter the dispensing, molecules within the first solution and the secondsolution do not substantially mix with each other, where the secondsolution includes a capture probe, and the dispensing results inattachment of the capture probe to the surface of the substrate.

In some embodiments, methods of generating a capture probe bound tosubstrate further include: curing the second solution, and removing thefirst solution from the substrate, where the cured second solution isretained on the surface of the substrate.

In some embodiments of methods of generating a capture probe bound tosubstrate, the surface of the substrate to which the capture probe isattached has an area that is smaller than a corresponding area generatedby dispensing the second solution onto the surface of the substrate inthe absence of the first solution. In some embodiments of methods ofgenerating a capture probe bound to substrate, the surface of thesubstrate to which the capture probe is attached has an area that isabout two-fold less than the corresponding area generated by dispensingthe second solution onto the surface of the substrate in the absence ofthe first solution.

In some embodiments, methods of generating a capture probe bound tosubstrate further include curing the first and second solutions on thesubstrate, thus generating a matrix. In some embodiments of methods ofgenerating a capture probe bound to substrate, the first and secondsolutions are cured by chemical means. In some embodiments of methods ofgenerating a capture probe bound to substrate, the first and secondsolutions are cured by photo-reactive means.

In some embodiments, methods of generating a capture probe bound tosubstrate further include shrinking the matrix, thus generating ashrunken matrix having an area to which the capture probe is attached,where the area in the shrunken matrix is reduced as compared to acorresponding area in the matrix.

In some embodiments, methods of generating a capture probe bound tosubstrate further include prior to the step of dispensing the secondsolution: curing the first solution to form a first solution matrix,where the first solution matrix can be reversibly expanded along one ormore of its axes. In some embodiments of methods of generating a captureprobe bound to substrate, the step of dispensing the second solutiononto the portion of the substrate including dispensing the secondsolution onto a surface of the first solution matrix in an expandedstate. In some embodiments, methods of generating a capture probe boundto substrate further includes, after the dispensing step: shrinking thefirst solution matrix along the one or more axes of the surface of thesubstrate.

In some embodiments of methods of generating a capture probe bound tosubstrate, the first solution has a viscosity that is about 0.1x-fold,0.2x-fold, 0.3x-fold, 0.4x-fold, 0.5x-fold, 0.6x-fold, 0.7x-fold,0.8x-fold, 0.9x-fold, 1.0x-fold, 1.1x-fold, 1.2x-fold, 1.3x-fold,1.4-fold, 1.5x-fold, 1.6x-fold, 1.7x-fold, 1.8x-fold, 1.9x-fold,2.0x-fold, 2.5x-fold, 3.0x-fold, 4.0x-fold, 5.0x fold, 6.0x-fold,7.0x-fold, 8.0x fold, 9.0x-fold or 10x-fold greater than the viscosityof the second solution.

In some embodiments of methods of generating a capture probe bound tosubstrate, the first solution has a lower nucleic acid diffusivity ascompared to the second solution.

In some embodiments of methods of generating a capture probe bound tosubstrate, the capture probe includes a spatial barcode. In someembodiments, the capture probe includes a unique molecular identifier.In some embodiments of methods of generating a capture probe bound tosubstrate, the capture probe includes a functional domain. In someembodiments of methods of generating a capture probe bound to substrate,the capture probe includes a cleavage domain, a spatial barcode, aunique molecular identifier, a functional domain, and a capture domain.

In another aspect, provided herein are methods of generating an array ofcapture probes bound to a substrate including dispensing a droplet of asecond solution onto a portion of a surface of a substrate coated with afirst solution, where after the dispensing, molecules within the firstsolution and the droplet of the second solution do not substantially mixwith each other, where the second solution includes a capture probe, andthe dispensing results in attachment of the capture probe to the surfaceof the substrate.

In some embodiments, methods of generating an array of capture probesbound to a substrate further include: curing the second solution, andremoving the first solution from the substrate, where the cured secondsolution is retained on the surface of the substrate.

In some embodiments of methods of generating an array of capture probesbound to a substrate, the surface of the substrate to which the captureprobe is attached has an area that is smaller than a corresponding areagenerated by dispensing the second solution onto the surface of thesubstrate in the absence of the first solution. In some embodiments ofmethods of generating an array of capture probes bound to a substrate,the surface of the substrate to which the capture probe is attached hasan area that is about two-fold less than the corresponding areagenerated by dispensing the second solution onto the surface of thesubstrate in the absence of the first solution.

In some embodiments, methods of generating an array of capture probesbound to a substrate further include curing the first and secondsolutions on the substrate, thus generating a matrix. In someembodiments of methods of generating an array of capture probes bound toa substrate, the first solution and the second solutions are cured bychemical means. In some embodiments of methods of generating an array ofcapture probes bound to a substrate, the first and second solutions arecured by photo-reactive means.

In some embodiments, methods of generating an array of capture probesbound to a substrate further include shrinking the matrix, thusgenerating a shrunken matrix having an area to which the capture probeis attached, where the area in the shrunken matrix is reduced ascompared to a corresponding area in the matrix.

In some embodiments, methods of generating an array of capture probesbound to a substrate further include, prior to the step of dispensingthe second solution: curing the first solution to form a first solutionmatrix, where the first solution matrix can be reversibly expanded alongone or more of its axes. In some embodiments of methods of generating anarray of capture probes bound to a substrate, the step of dispensing thesecond solution onto the portion of the substrate including dispensingthe second solution onto a surface of the first solution matrix in anexpanded state.

In some embodiments, methods of generating an array of capture probesbound to a substrate further include after the dispensing step:shrinking the first solution matrix along the one or more axes of thesurface of the substrate.

In some embodiments of methods of generating an array of capture probesbound to a substrate, the first solution has a viscosity that is about0.1x-fold, 0.2x-fold, 0.3x-fold, 0.4x-fold, 0.5x-fold, 0.6x-fold,0.7x-fold, 0.8x-fold, 0.9x-fold, 1.0x-fold, 1.1x-fold, 1.2x-fold,1.3x-fold, 1.4-fold, 1.5x-fold, 1.6x-fold, 1.7x-fold, 1.8x-fold,1.9x-fold, 2.0x-fold, 2.5x-fold, 3.0x-fold, 4.0x-fold, 5.0x fold,6.0x-fold, 7.0x-fold, 8.0x fold, 9.0x-fold or 10x-fold greater than theviscosity of the second solution.

In some embodiments of methods of generating an array of capture probesbound to a substrate, the first solution has a lower nucleic aciddiffusivity as compared to the second solution.

In some embodiments of methods of generating an array of capture probesbound to a substrate, the capture probe includes a spatial barcode. Insome embodiments of methods of generating an array of capture probesbound to a substrate, the at least one of the capture probes includes aunique molecular identifier. In some embodiments of methods ofgenerating an array of capture probes bound to a substrate, the captureprobe includes a functional domain. In some embodiments of methods ofgenerating an array of capture probes bound to a substrate, the captureprobe includes a cleavage domain, a spatial barcode, a unique molecularidentifier, a functional domain, and a capture domain.

In another aspect, provided herein are methods for spatially profilingan analyte in a biological sample including (a) dispensing a droplet ofa second solution onto a portion of a surface of a substrate coated witha the first solution, where after the dispensing, molecules within thefirst solution and the droplet of the second solution do notsubstantially mix with each other, where the second solution includes acapture probe, and the dispensing results in attachment of the captureprobe to the surface of the substrate, (b) contacting the biologicalsample with the spatial array such that the analyte present in thebiological sample is captured by the capture probes, thereby determiningthe spatial profile of the captured analyte.

In some embodiments of methods for spatially profiling an analyte in abiological sample, at least one of the capture probes includes acleavage domain, a spatial barcode, a unique molecular identifier, afunctional domain, and a capture domain.

All publications, patents, patent applications, and informationavailable on the internet and mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication, patent, patent application, or item of information wasspecifically and individually indicated to be incorporated by reference.To the extent publications, patents, patent applications, and items ofinformation incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

Where values are described in terms of ranges, it should be understoodthat the description includes the disclosure of all possible sub-rangeswithin such ranges, as well as specific numerical values that fallwithin such ranges irrespective of whether a specific numerical value orspecific sub-range is expressly stated.

The term “each,” when used in reference to a collection of items, isintended to identify an individual item in the collection but does notnecessarily refer to every item in the collection, unless expresslystated otherwise, or unless the context of the usage clearly indicatesotherwise.

Various embodiments of the features of this disclosure are describedherein. However, it should be understood that such embodiments areprovided merely by way of example, and numerous variations, changes, andsubstitutions can occur to those skilled in the art without departingfrom the scope of this disclosure. It should also be understood thatvarious alternatives to the specific embodiments described herein arealso within the scope of this disclosure.

DESCRIPTION OF DRAWINGS

The following drawings illustrate certain embodiments of the featuresand advantages of this disclosure. These embodiments are not intended tolimit the scope of the appended claims in any manner. Like referencesymbols in the drawings indicate like elements.

FIG. 1 shows an exemplary spatial analysis workflow.

FIG. 2 shows an exemplary spatial analysis workflow.

FIG. 3 shows an exemplary spatial analysis workflow.

FIG. 4 shows an exemplary spatial analysis workflow.

FIG. 5 shows an exemplary spatial analysis workflow.

FIG. 6 is a schematic diagram showing an example of a barcoded captureprobe, as described herein.

FIG. 7 is a schematic illustrating a cleavable capture probe, whereinthe cleaved capture probe can enter into a non-permeabilized cell andbind to target analytes within the sample.

FIG. 8 is a schematic diagram of an exemplary multiplexedspatially-barcoded feature.

FIG. 9 is a schematic diagram of an exemplary analyte capture agent.

FIG. 10 is a schematic diagram depicting an exemplary interactionbetween a feature-immobilized capture probe 1024 and an analyte captureagent 1026.

FIGS. 11A, 11B, and 11C are schematics illustrating how streptavidincell tags can be utilized in an array-based system to produce aspatially-barcoded cells or cellular contents.

FIG. 12 is a schematic showing the arrangement of barcoded featureswithin an array.

FIG. 13 is a schematic illustrating a side view of a diffusion-resistantmedium, e.g., a lid.

FIGS. 14A and 14B are schematics illustrating expanded FIG. 14A and sideviews FIG. 14B of an electrophoretic transfer system configured todirect transcript analytes toward a spatially-barcoded capture probearray.

FIG. 15 is a schematic illustrating an exemplary workflow protocolutilizing an electrophoretic transfer system.

FIG. 16 shows an example of a microfluidic channel structure 1600 forpartitioning dissociated sample (e.g., biological particles orindividual cells from a sample).

FIG. 17A shows an example of a microfluidic channel structure 1700 fordelivering spatial barcode carrying beads to droplets.

FIG. 17B shows a cross-section view of another example of a microfluidicchannel structure 1750 with a geometric feature for controlledpartitioning.

FIG. 17C shows an example of a workflow schematic.

FIG. 18 is a schematic depicting cell tagging using either covalentconjugation of the analyte binding moiety to the cell surface ornon-covalent interactions with cell membrane elements.

FIG. 19 is a schematic depicting cell tagging using eithercell-penetrating peptides or delivery systems.

FIG. 20A is a workflow schematic illustrating exemplary, non-limiting,non-exhaustive steps for “pixelating” a sample, wherein the sample iscut, stamped, microdissected, or transferred by hollow-needle ormicroneedle, moving a small portion of the sample into an individualpartition or well.

FIG. 20B is a schematic depicting multi-needle pixilation, wherein anarray of needles punched through a sample on a scaffold and intonanowells containing gel beads and reagents below. Once the needle is inthe nanowell, the cell(s) are ejected.

FIG. 21 shows a workflow schematic illustrating exemplary, non-limiting,non-exhaustive steps for dissociating a spatially-barcoded sample foranalysis via droplet or flow cell analysis methods.

FIG. 22A is a schematic diagram showing an example sample handlingapparatus that can be used to implement various steps and methodsdescribed herein.

FIG. 22B is a schematic diagram showing an example imaging apparatusthat can be used to obtain images of biological samples, analytes, andarrays of features.

FIG. 22C is a schematic diagram of an example of a control unit of theapparatus of FIGS. 22A and 22B.

FIG. 23A shows a histological section of an invasive ductal carcinomaannotated by a pathologist.

FIG. 23B shows a tissue plot with spots colored by unsupervisedclustering.

FIG. 23C is a tSNE plot of spots colored by unsupervised clustering.

FIG. 23D shows a gene expression heat map of the most variable genesbetween 9 clusters.

FIG. 23E shows the expression levels of genes corresponding to humanepidermal growth factor receptor 2 (Her2), estrogen receptor (ER), andprogesterone receptor (PR) in the tissue section.

FIG. 23F shows the expression levels of genes of top differentiallyexpressed genes from each of the 9 clusters on individual plots.

FIG. 23G shows the expression levels of genes of top differentiallyexpressed genes from each of the 9 clusters on a single plot.

FIG. 23H is a plot of the expression levels of the top differentiallyexpressed genes from each of the 9 clusters in invasive ductal cellcarcinoma (IDC) and normal breast tissue.

FIG. 23I shows the expression of KRT14 in IDC and match normal tissue.

FIG. 23J is a plot of the expression levels of extracellular matrixgenes in IDC and normal tissue.

FIG. 24 is a schematic diagram showing a method for generating captureprobes of an array for spatial analysis of analytes in biologicalsample.

FIG. 25 is a schematic diagram showing a method of capturing an analytewith a capture domain and performing reverse transcription to obtain acomplementary cDNA sequence.

FIG. 26 shows a schematic depicting steps involved in generating ahigh-density spatially barcoded array with increased resolution.

FIG. 27 is a schematic diagram showing an exemplary two-step barcodeconstruction process to build an array.

FIG. 28 is a schematic diagram showing an exemplary two-step barcodeconstruction process to build an array.

FIG. 29 is a schematic diagram showing an exemplary spatial arraypreparation process.

FIG. 30 is a schematic diagram showing an exemplary design for a fluidicisolation chamber.

FIGS. 31A-C depict an illustrative embodiment of the methods forpreparing a spatial array of the present disclosure. FIG. 31A depicts asubstrate provided with first oligonucleotides attached thereto in a5′-to-3′ orientation, wherein the phosphodiester and exocyclic aminegroups are protected. FIG. 31B depicts 5′-to-3 light-directed spatialsynthesis of second oligonucleotides to the free 3′-ends of the firstoligonucleotide on the substrate, wherein the second oligonucleotidehave different sequences in the two different regions. FIG. 31C depictsligation of a third oligonucleotide to the free 3′-ends of the twodifferent second oligonucleotides.

FIGS. 32A-C depict an illustrative embodiment of the methods forpreparing a spatial array of the present disclosure. FIG. 32A depicts exsitu solid phase synthesis of a first oligonucleotide in 3′-to-5′direction followed by cleavage from the feature with the phosphodiesterand exocyclic amine groups protected. FIG. 32B depicts coupling of theex situ synthesized first oligonucleotide to a substrate in a 5′-to-3′orientation. FIG. 32C depicts 5′-to-3′ light-directed spatial synthesisof second oligonucleotides to the free 3′-ends of the firstoligonucleotide on the substrate, wherein the second oligonucleotideshave different sequences in the two different regions.

FIGS. 33A-B depict an illustrative embodiment of the methods forpreparing a spatial array of the present disclosure. FIG. 33A depicts afirst oligonucleotide with phosphodiester and exocyclic amine protectinggroups attached to a substrate in a 5′-to-3′ orientation, wherein thefirst oligonucleotide has been in situ synthesized on the surface of thesubstrate. FIG. 33B depicts 5′-to-3 light-directed spatial synthesis ofsecond oligonucleotides to the free 3′-ends of the first oligonucleotide(with first segment phosphodiester and exocyclic amine protecting groupsin place) on the substrate, wherein the second oligonucleotides havedifferent sequences in the two different regions.

FIGS. 34A-B depict an illustrative embodiment of the methods forpreparing a spatial array of the present disclosure. FIG. 34A depicts afirst oligonucleotide phosphodiester and exocyclic amine protectinggroups attached to a substrate in a 5′-to-3′ orientation, wherein thefirst oligonucleotide has been synthesized on the surface of thesubstrate using 5′-to-3′ light-directed spatial synthesis. FIG. 34Bdepicts 5′-to-3 light-directed spatial synthesis of secondoligonucleotides to the free 3′-ends of the first oligonucleotide (withfirst oligonucleotide phosphodiester and exocyclic amine protectinggroups in place) on the substrate, wherein the second oligonucleotideshave different sequences in the two different regions.

FIGS. 35A-C depict an illustrative embodiment of the methods forpreparing a spatial array of the present disclosure. FIG. 35A depictscoupling of an ex situ synthesized first oligonucleotide in 3′-to-5′direction without phosphodiester and exocyclic amine groups protected.FIG. 35B depicts in situ protection of the phosphodiester and exocyclicamine groups of the first oligonucleotide. FIG. 35C depicts 5′-to-3light-directed spatial synthesis of second oligonucleotides to the free3′-ends of the first oligonucleotide the substrate, wherein the secondoligonucleotides have different sequences in the two different regions.

FIGS. 36A-C depict an illustrative embodiment of the methods forpreparing a spatial array of the present disclosure. FIG. 36A depictscoupling of an ex situ synthesized first oligonucleotide in 3′-to-5′direction without phosphodiester and exocyclic amine groups protected.FIG. 36B depicts in situ hybridization of a complementaryoligonucleotide to the first oligonucleotide, whereby the phosphodiesterand exocyclic amine groups of the first oligonucleotide are protected.FIG. 36C depicts 5′-to-3 light-directed spatial synthesis of secondoligonucleotides to the free 3′-ends of the first oligonucleotide on thesubstrate while the first oligonucleotide is hybridized to acomplementary oligonucleotide, and wherein the synthesized secondoligonucleotides have different sequences in the two different regions.

FIG. 37 shows an example of a spatially hierarchical spatial array, inwhich one region is divided into nine subregions, which is furtherdivided into fifteen sub-sub regions. Each spatial barcode of the 135sub-sub regions includes a region spatial barcode, a sub region spatialbarcode, and a sub-sub region spatial barcode.

FIG. 38 shows an example of a library structure and amplification schemeincluding a two level spatial barcode, wherein the spatial barcodeincludes a region spatial barcode prefix and a specific spatial barcodesuffix.

FIG. 39 is a workflow schematic showing exemplary, non-limiting,non-exhaustive steps for preparing a spatial array and decoding theclusters on the slide.

FIG. 40 is a workflow schematic showing oligonucleotide construction.

FIG. 41 shows a method for generating a capture probe.

FIG. 42 is a graph showing detection of cDNA with ligated and unligatedpolyT and gene specific, e.g., Penk gene, capture domains.

FIG. 43 is a fluorescent image showing detection of Penk mRNA with aPenk specific capture probe generated by the method shown in FIG. 41compared to a control tissue optimization slide.

FIG. 44 shows an exemplary ligation-based method for generating acapture probe using two ligation handles.

DETAILED DESCRIPTION I. Introduction

This disclosure describes apparatus, systems, methods, and compositionsfor spatial analysis of biological samples. This section describescertain general terminology, analytes, sample types, and preparativesteps that are referred to in later sections of the disclosure.

(A) Spatial Analysis

Tissues and cells can be obtained from any source. For example, tissuesand cells can be obtained from single-cell or multicellular organisms(e.g., a mammal). Tissues and cells obtained from a mammal, e.g., ahuman, often have varied analyte levels (e.g., gene and/or proteinexpression) which can result in differences in cell morphology and/orfunction. The position of a cell within a tissue can affect, e.g., thecell’s fate, behavior, morphology, and signaling and cross-talk withother cells in the tissue. Information regarding the differences inanalyte levels (gene and/or protein expression) within different cellsin a tissue of a mammal can also help physicians select or administer atreatment that will be effective in the single-cell or multicellularorganisms (e.g., a mammal) based on the detected differences in analytelevels within different cells in the tissue. Differences in analytelevels within different cells in a tissue of a mammal can also provideinformation on how tissues (e.g., healthy and diseased tissues) functionand/or develop. Differences in analyte levels within different cells ina tissue of a mammal can also provide information of differentmechanisms of disease pathogenesis in a tissue and mechanism of actionof a therapeutic treatment within a tissue. Differences in analytelevels within different cells in a tissue of a mammal can also provideinformation on drug resistance mechanisms and the development of thesame in a tissue of a mammal. Differences in the presence or absence ofanalytes within different cells in a tissue of a multicellular organism(e.g., a mammal) can provide information on drug resistance mechanismsand the development of the same in a tissue of a multicellular organism.

The spatial analysis methodologies herein provide for the detection ofdifferences in an analyte level (e.g., gene and/or protein expression)within different cells in a tissue of a mammal or within a single cellfrom a mammal. For example, spatial analysis methodologies can be usedto detect the differences in analyte levels (e.g., gene and/or proteinexpression) within different cells in histological slide samples, thedata from which can be reassembled to generate a three-dimensional mapof analyte levels (e.g., gene and/or protein expression) of a tissuesample obtained from a mammal, e.g., with a degree of spatial resolution(e.g., single-cell resolution).

Spatial heterogeneity in developing systems has typically been studiedvia RNA hybridization, immunohistochemistry, fluorescent reporters, orpurification or induction of pre-defined subpopulations and subsequentgenomic profiling (e.g., RNA-seq). Such approaches, however, rely on arelatively small set of pre-defined markers, therefore introducingselection bias that limits discovery. These prior approaches also relyon a priori knowledge. Spatial RNA assays traditionally relied onstaining for a limited number of RNA species. In contrast, single-cellRNA-sequencing allows for deep profiling of cellular gene expression(including non-coding RNA), but the established methods separate cellsfrom their native spatial context.

Current spatial analysis methodologies provide a vast amount of analytelevel and/or expression data for a variety of multiple analytes within asample at high spatial resolution, e.g., while retaining the nativespatial context. Spatial analysis methods include, e.g., the use of acapture probe including a spatial barcode (e.g., a nucleic acid sequencethat provides information as to the position of the capture probe withina cell or a tissue sample (e.g., mammalian cell or a mammalian tissuesample) and a capture domain that is capable of binding to an analyte(e.g., a protein and/or nucleic acid) produced by and/or present in acell. As described herein, the spatial barcode can be a nucleic acidthat has a unique sequence, a unique fluorophore or a unique combinationof fluorophores, a unique amino acid sequence, a unique heavy metal or aunique combination of heavy metals, or any other unique detectableagent. The capture domain can be any agent that is capable of binding toan analyte produced by and/or present in a cell (e.g., a nucleic acidthat is capable of hybridizing to a nucleic acid from a cell (e.g., anmRNA, genomic DNA, mitochondrial DNA, or miRNA), a substrate includingan analyte, a binding partner of an analyte, or an antibody that bindsspecifically to an analyte). A capture probe can also include a nucleicacid sequence that is complementary to a sequence of a universal forwardand/or universal reverse primer. A capture probe can also include acleavage site (e.g., a cleavage recognition site of a restrictionendonuclease), a photolabile bond, a thermosensitive bond, or achemical-sensitive bond.

The binding of an analyte to a capture probe can be detected using anumber of different methods, e.g., nucleic acid sequencing, fluorophoredetection, nucleic acid amplification, detection of nucleic acidligation, and/or detection of nucleic acid cleavage products. In someexamples, the detection is used to associate a specific spatial barcodewith a specific analyte produced by and/or present in a cell (e.g., amammalian cell).

Capture probes can be, e.g., attached to a surface, e.g., a solid array,a bead, or a coverslip. In some examples, capture probes are notattached to a surface. In some examples, capture probes can beencapsulated within, embedded within, or layered on a surface of apermeable composition (e.g., any of the substrates described herein).For example, capture probes can be encapsulated or disposed within apermeable bead (e.g., a gel bead). In some examples, capture probes canbe encapsulated within, embedded within, or layered on a surface of asubstrate (e.g., any of the exemplary substrates described herein, suchas a hydrogel or a porous membrane).

In some examples, a cell or a tissue sample including a cell arecontacted with capture probes attached to a substrate (e.g., a surfaceof a substrate), and the cell or tissue sample is permeabilized to allowanalytes to be released from the cell and bind to the capture probesattached to the substrate. In some examples, analytes released from acell can be actively directed to the capture probes attached to asubstrate using a variety of methods, e.g., electrophoresis, chemicalgradient, pressure gradient, fluid flow, or magnetic field.

In other examples, a capture probe can be directed to interact with acell or a tissue sample using a variety of methods, e.g., inclusion of alipid anchoring agent in the capture probe, inclusion of an agent thatbinds specifically to, or forms a covalent bond with a membrane proteinin the capture probe, fluid flow, pressure gradient, chemical gradient,or magnetic field.

Non-limiting aspects of spatial analysis methodologies are described inWO 2011/127099, WO 2014/210233, WO 2014/210225, WO 2016/162309, WO2018/091676, WO 2012/140224, WO 2014/060483, U.S. Pat. No. 10,002,316,U.S. Pat. No. 9,727,810, U.S. Pat. Application Publication No.2017/0016053, Rodriques et al., Science 363(6434):1463-1467, 2019; WO2018/045186, Lee et al., Nat. Protoc. 10(3):442-458, 2015; WO2016/007839, WO 2018/045181, WO 2014/163886, Trejo et al., PLoS ONE14(2):e0212031, 2019, U.S. Pat. Application Publication No.2018/0245142, Chen et al., Science 348(6233):aaa6090, 2015, Gao et al.,BMC Biol. 15:50, 2017, WO 2017/144338, WO 2018/107054, WO 2017/222453,WO 2019/068880, WO 2011/094669, U.S. Pat. No. 7,709,198, U.S. Pat. No.8,604,182, U.S. Pat. No. 8,951,726, U.S. Pat. No. 9,783,841, U.S. Pat.No. 10,041,949, WO 2016/057552, WO 2017/147483, WO 2018/022809, WO2016/166128, WO 2017/027367, WO 2017/027368, WO 2018/136856, WO2019/075091, U.S. Pat. No. 10,059,990, WO 2018/057999, WO 2015/161173,and Gupta et al., Nature Biotechnol. 36:1197-1202, 2018, and can be usedherein in any combination. Further non-limiting aspects of spatialanalysis methodologies are described herein.

(B) General Terminology

Specific terminology is used throughout this disclosure to explainvarious aspects of the apparatus, systems, methods, and compositionsthat are described. This sub-section includes explanations of certainterms that appear in later sections of the disclosure. To the extentthat the descriptions in this section are in apparent conflict withusage in other sections of this disclosure, the definitions in thissection will control.

(I) Barcode

A “barcode” is a label, or identifier, that conveys or is capable ofconveying information (e.g., information about an analyte in a sample, abead, and/or a capture probe). A barcode can be part of an analyte, orindependent of an analyte. A barcode can be attached to an analyte. Aparticular barcode can be unique relative to other barcodes.

Barcodes can have a variety of different formats. For example, barcodescan include polynucleotide barcodes, random nucleic acid and/or aminoacid sequences, and synthetic nucleic acid and/or amino acid sequences.A barcode can be attached to an analyte or to another moiety orstructure in a reversible or irreversible manner. A barcode can be addedto, for example, a fragment of a deoxyribonucleic acid (DNA) orribonucleic acid (RNA) sample before or during sequencing of the sample.Barcodes can allow for identification and/or quantification ofindividual sequencing-reads (e.g., a barcode can be or can include aunique molecular identifier or “UMI”).

Barcodes can spatially-resolve molecular components found in biologicalsamples, for example, at single-cell resolution (e.g., a barcode can beor can include a “spatial barcode”). In some embodiments, a barcodeincludes both a UMI and a spatial barcode. In some embodiments, abarcode includes two or more sub-barcodes that together function as asingle barcode. For example, a polynucleotide barcode can include two ormore polynucleotide sequences (e.g., sub-barcodes) that are separated byone or more non-barcode sequences.

(II) Nucleic Acid and Nucleotide

The terms “nucleic acid” and “nucleotide” are intended to be consistentwith their use in the art and to include naturally-occurring species orfunctional analogs thereof. Particularly useful functional analogs ofnucleic acids are capable of hybridizing to a nucleic acid in asequence-specific fashion (e.g., capable of hybridizing to two nucleicacids such that ligation can occur between the two hybridized nucleicacids) or are capable of being used as a template for replication of aparticular nucleotide sequence. Naturally-occurring nucleic acidsgenerally have a backbone containing phosphodiester bonds. An analogstructure can have an alternate backbone linkage including any of avariety of those known in the art. Naturally-occurring nucleic acidsgenerally have a deoxyribose sugar (e.g., found in deoxyribonucleic acid(DNA)) or a ribose sugar (e.g., found in ribonucleic acid (RNA)).

A nucleic acid can contain nucleotides having any of a variety ofanalogs of these sugar moieties that are known in the art. A nucleicacid can include native or non-native nucleotides. In this regard, anative deoxyribonucleic acid can have one or more bases selected fromthe group consisting of adenine (A), thymine (T), cytosine (C), orguanine (G), and a ribonucleic acid can have one or more bases selectedfrom the group consisting of uracil (U), adenine (A), cytosine (C), orguanine (G). Useful non-native bases that can be included in a nucleicacid or nucleotide are known in the art.

(III) Probe and Target

A “probe” or a “target,” when used in reference to a nucleic acid orsequence of a nucleic acids, is intended as a semantic identifier forthe nucleic acid or sequence in the context of a method or composition,and does not limit the structure or function of the nucleic acid orsequence beyond what is expressly indicated.

(IV) Oligonucleotide and Polynucleotide

The terms “oligonucleotide” and “polynucleotide” are usedinterchangeably to refer to a single-stranded multimer of nucleotidesfrom about 2 to about 500 nucleotides in length. Oligonucleotides can besynthetic, made enzymatically (e.g., via polymerization), or using a“split-pool” method. Oligonucleotides can include ribonucleotidemonomers (i.e., can be oligoribonucleotides) and/or deoxyribonucleotidemonomers (i.e., oligodeoxyribonucleotides). In some examples,oligonucleotides can include a combination of both deoxyribonucleotidemonomers and ribonucleotide monomers in the oligonucleotide (e.g.,random or ordered combination of deoxyribonucleotide monomers andribonucleotide monomers). An oligonucleotide can be 4 to 10, 10 to 20,21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100,100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400,or 400-500 nucleotides in length, for example. Oligonucleotides caninclude one or more functional moieties that are attached (e.g.,covalently or non-covalently) to the multimer structure. For example, anoligonucleotide can include one or more detectable labels (e.g., aradioisotope or fluorophore).

(V) Subject

A “subject” is an animal, such as a mammal (e.g., human or a non-humansimian), or avian (e.g., bird), or other organism, such as a plant.Examples of subjects include, but are not limited to, a mammal such as arodent, mouse, rat, rabbit, guinea pig, ungulate, horse, sheep, pig,goat, cow, cat, dog, primate (i.e. human or non-human primate); a plantsuch as Arabidopsis thaliana, corn, sorghum, oat, wheat, rice, canola,or soybean; an algae such as Chlamydomonas reinhardtii; a nematode suchas Caenorhabditis elegans; an insect such as Drosophila melanogaster,mosquito, fruit fly, or honey bee; an arachnid such as a spider; a fishsuch as zebrafish; a reptile; an amphibian such as a frog or Xenopuslaevis; a Dictyostelium discoideum; a fungi such as Pneumocystiscarinii, Takifugu rubripes, yeast, Saccharamoyces cerevisiae orSchizosaccharomyces pombe; or a Plasmodium falciparum.

(VI) Genome

A “genome” generally refers to genomic information from a subject, whichcan be, for example, at least a portion of, or the entirety of, thesubject’s gene-encoded hereditary information. A genome can includecoding regions (e.g., that code for proteins) as well as non-codingregions. A genome can include the sequences of some or all of thesubject’s chromosomes. For example, the human genome ordinarily has atotal of 46 chromosomes. The sequences of some or all of these canconstitute the genome.

(VII) Adaptor, Adapter, and Tag

An “adaptor,” an “adapter,” and a “tag” are terms that are usedinterchangeably in this disclosure, and refer to species that can becoupled to a polynucleotide sequence (in a process referred to as“tagging”) using any one of many different techniques including (but notlimited to) ligation, hybridization, and tagmentation. Adaptors can alsobe nucleic acid sequences that add a function, e.g., spacer sequences,primer sequences/sites, barcode sequences, unique molecular identifiersequences.

(VIII) Hybridizing, Hybridize, Annealing, and Anneal

The terms “hybridizing,” “hybridize,” “annealing,” and “anneal” are usedinterchangeably in this disclosure, and refer to the pairing ofsubstantially complementary or complementary nucleic acid sequenceswithin two different molecules. Pairing can be achieved by any processin which a nucleic acid sequence joins with a substantially or fullycomplementary sequence through base pairing to form a hybridizationcomplex. For purposes of hybridization, two nucleic acid sequences are“substantially complementary” if at least 60% (e.g., at least 70%, atleast 80%, or at least 90%) of their individual bases are complementaryto one another.

(IX) Primer

A “primer” is a single-stranded nucleic acid sequence having a 3′ endthat can be used as a chemical substrate for a nucleic acid polymerasein a nucleic acid extension reaction. RNA primers are formed of RNAnucleotides, and are used in RNA synthesis, while DNA primers are formedof DNA nucleotides and used in DNA synthesis. Primers can also includeboth RNA nucleotides and DNA nucleotides (e.g., in a random or designedpattern). Primers can also include other natural or syntheticnucleotides described herein that can have additional functionality. Insome examples, DNA primers can be used to prime RNA synthesis and viceversa (e.g., RNA primers can be used to prime DNA synthesis). Primerscan vary in length. For example, primers can be about 6 bases to about120 bases. For example, primers can include up to about 25 bases.

(X) Primer Extension

A “primer extension” refers to any method where two nucleic acidsequences (e.g., a constant region from each of two distinct captureprobes) become linked (e.g., hybridized) by an overlap of theirrespective terminal complementary nucleic acid sequences (i.e., forexample, 3′ termini). Such linking can be followed by nucleic acidextension (e.g., an enzymatic extension) of one, or both termini usingthe other nucleic acid sequence as a template for extension. Enzymaticextension can be performed by an enzyme including, but not limited to, apolymerase and/or a reverse transcriptase.

(XI) Proximity Ligation

A “proximity ligation” is a method of ligating two (or more) nucleicacid sequences that are in proximity with each other through enzymaticmeans (e.g., a ligase). In some embodiments, proximity ligation caninclude a “gap-filling” step that involves incorporation of one or morenucleic acids by a polymerase, based on the nucleic acid sequence of atemplate nucleic acid molecule, spanning a distance between the twonucleic acid molecules of interest (see, e.g., U.S. Pat. No. 7,264,929,the entire contents of which are incorporated herein by reference).

A wide variety of different methods can be used for proximity ligatingnucleic acid molecules, including (but not limited to) “sticky-end” and“blunt-end” ligations. Additionally, single-stranded ligation can beused to perform proximity ligation on a single-stranded nucleic acidmolecule. Sticky-end proximity ligations involve the hybridization ofcomplementary single-stranded sequences between the two nucleic acidmolecules to be joined, prior to the ligation event itself. Blunt-endproximity ligations generally do not include hybridization ofcomplementary regions from each nucleic acid molecule because bothnucleic acid molecules lack a single-stranded overhang at the site ofligation.

(XII) Nucleic Acid Extension

A “nucleic acid extension” generally involves incorporation of one ormore nucleic acids (e.g., A, G, C, T, U, nucleotide analogs, orderivatives thereof) into a molecule (such as, but not limited to, anucleic acid sequence) in a template-dependent manner, such thatconsecutive nucleic acids are incorporated by an enzyme (such as apolymerase or reverse transcriptase), thereby generating a newlysynthesized nucleic acid molecule. For example, a primer that hybridizesto a complementary nucleic acid sequence can be used to synthesize a newnucleic acid molecule by using the complementary nucleic acid sequenceas a template for nucleic acid synthesis. Similarly, a 3′ polyadenylatedtail of an mRNA transcript that hybridizes to a poly (dT) sequence(e.g., capture domain) can be used as a template for single-strandsynthesis of a corresponding cDNA molecule.

(XIII) PCR Amplification

A “PCR amplification” refers to the use of a polymerase chain reaction(PCR) to generate copies of genetic material, including DNA and RNAsequences. Suitable reagents and conditions for implementing PCR aredescribed, for example, in U.S. Pats. 4,683,202, 4,683,195, 4,800,159,4,965,188, and 5,512,462, the entire contents of each of which areincorporated herein by reference. In a typical PCR amplification, thereaction mixture includes the genetic material to be amplified, anenzyme, one or more primers that are employed in a primer extensionreaction, and reagents for the reaction. The oligonucleotide primers areof sufficient length to provide for hybridization to complementarygenetic material under annealing conditions. The length of the primersgenerally depends on the length of the amplification domains, but willtypically be at least 4 bases, at least 5 bases, at least 6 bases, atleast 8 bases, at least 9 bases, at least 10 base pairs (bp), at least11 bp, at least 12 bp, at least 13 bp, at least 14 bp, at least 15 bp,at least 16 bp, at least 17 bp, at least 18 bp, at least 19 bp, at least20 bp, at least 25 bp, at least 30 bp, at least 35 bp, and can be aslong as 40 bp or longer, where the length of the primers will generallyrange from 18 to 50 bp. The genetic material can be contacted with asingle primer or a set of two primers (forward and reverse primers),depending upon whether primer extension, linear or exponentialamplification of the genetic material is desired.

In some embodiments, the PCR amplification process uses a DNA polymeraseenzyme. The DNA polymerase activity can be provided by one or moredistinct DNA polymerase enzymes. In certain embodiments, the DNApolymerase enzyme is from a bacterium, e.g., the DNA polymerase enzymeis a bacterial DNA polymerase enzyme. For instance, the DNA polymerasecan be from a bacterium of the genus Escherichia, Bacillus,Thermophilus, or Pyrococcus.

Suitable examples of DNA polymerases that can be used include, but arenot limited to: E.coli DNA polymerase I, Bsu DNA polymerase, Bst DNApolymerase, Taq DNA polymerase, VENT™ DNA polymerase, DEEPVENT™ DNApolymerase, LongAmp® Taq DNA polymerase, LongAmp® Hot Start Taq DNApolymerase, Crimson LongAmp® Taq DNA polymerase, Crimson Taq DNApolymerase, OneTaq® DNA polymerase, OneTaq® Quick-Load® DNA polymerase,Hemo KlenTaq® DNA polymerase, REDTaq® DNA polymerase, Phusion® DNApolymerase, Phusion® High-Fidelity DNA polymerase, Platinum Pfx DNApolymerase, AccuPrime Pfx DNA polymerase, Phi29 DNA polymerase, Klenowfragment, Pwo DNA polymerase, Pfu DNA polymerase, T4 DNA polymerase andT7 DNA polymerase enzymes.

The term “DNA polymerase” includes not only naturally-occurring enzymesbut also all modified derivatives thereof, including also derivatives ofnaturally-occurring DNA polymerase enzymes. For instance, in someembodiments, the DNA polymerase can have been modified to remove 5′-3′exonuclease activity. Sequence-modified derivatives or mutants of DNApolymerase enzymes that can be used include, but are not limited to,mutants that retain at least some of the functional, e.g., DNApolymerase activity of the wild-type sequence. Mutations can affect theactivity profile of the enzymes, e.g., enhance or reduce the rate ofpolymerization, under different reaction conditions, e.g., temperature,template concentration, primer concentration, etc. Mutations orsequence-modifications can also affect the exonuclease activity and/orthermostability of the enzyme.

In some embodiments, PCR amplification can include reactions such as,but not limited to, a strand-displacement amplification reaction, arolling circle amplification reaction, a ligase chain reaction, atranscription-mediated amplification reaction, an isothermalamplification reaction, and/or a loop-mediated amplification reaction.

In some embodiments, PCR amplification uses a single primer that iscomplementary to the 3′ tag of target DNA fragments. In someembodiments, PCR amplification uses a first and a second primer, whereat least a 3′ end portion of the first primer is complementary to atleast a portion of the 3′ tag of the target nucleic acid fragments, andwhere at least a 3′ end portion of the second primer exhibits thesequence of at least a portion of the 5′ tag of the target nucleic acidfragments. In some embodiments, a 5′ end portion of the first primer isnon-complementary to the 3′ tag of the target nucleic acid fragments,and a 5′ end portion of the second primer does not exhibit the sequenceof at least a portion of the 5′ tag of the target nucleic acidfragments. In some embodiments, the first primer includes a firstuniversal sequence and/or the second primer includes a second universalsequence.

In some embodiments (e.g., when the PCR amplification amplifies capturedDNA), the PCR amplification products can be ligated to additionalsequences using a DNA ligase enzyme. The DNA ligase activity can beprovided by one or more distinct DNA ligase enzymes. In someembodiments, the DNA ligase enzyme is from a bacterium, e.g., the DNAligase enzyme is a bacterial DNA ligase enzyme. In some embodiments, theDNA ligase enzyme is from a virus (e.g., a bacteriophage). For instance,the DNA ligase can be T4 DNA ligase. Other enzymes appropriate for theligation step include, but are not limited to, Tth DNA ligase, Taq DNAligase, Thermococcus sp. (strain 9oN) DNA ligase (9oNTM DNA ligase,available from New England Biolabs, Ipswich, MA), and Ampligase™(available from Epicentre Biotechnologies, Madison, WI). Derivatives,e.g., sequence-modified derivatives, and/or mutants thereof, can also beused.

In some embodiments, genetic material is amplified by reversetranscription polymerase chain reaction (RT-PCR). The desired reversetranscriptase activity can be provided by one or more distinct reversetranscriptase enzymes, suitable examples of which include, but are notlimited to: M-MLV, MuLV, AMV, HIV, ArrayScript™, MultiScribe™,ThermoScript™, and SuperScript® I, II, III, and IV enzymes. “Reversetranscriptase” includes not only naturally occurring enzymes, but allsuch modified derivatives thereof, including also derivatives ofnaturally-occurring reverse transcriptase enzymes.

In addition, reverse transcription can be performed usingsequence-modified derivatives or mutants of M-MLV, MuLV, AMV, and HIVreverse transcriptase enzymes, including mutants that retain at leastsome of the functional, e.g., reverse transcriptase, activity of thewild-type sequence. The reverse transcriptase enzyme can be provided aspart of a composition that includes other components, e.g., stabilizingcomponents that enhance or improve the activity of the reversetranscriptase enzyme, such as RNase inhibitor(s), inhibitors ofDNA-dependent DNA synthesis, e.g., actinomycin D. Many sequence-modifiedderivative or mutants of reverse transcriptase enzymes, e.g., M-MLV, andcompositions including unmodified and modified enzymes are commerciallyavailable, e.g., ArrayScript™, MultiScribe™, ThermoScript™, andSuperScript® I, II, III, and IV enzymes.

Certain reverse transcriptase enzymes (e.g., Avian Myeloblastosis Virus(AMV) Reverse Transcriptase and Moloney Murine Leukemia Virus (M-MuLV,MMLV) Reverse Transcriptase) can synthesize a complementary DNA strandusing both RNA (cDNA synthesis) and single-stranded DNA (ssDNA) as atemplate. Thus, in some embodiments, the reverse transcription reactioncan use an enzyme (reverse transcriptase) that is capable of using bothRNA and ssDNA as the template for an extension reaction, e.g., an AMV orMMLV reverse transcriptase.

In some embodiments, the quantification of RNA and/or DNA is carried outby real-time PCR (also known as quantitative PCR or qPCR), usingtechniques well known in the art, such as but not limited to “TAQMAN™”or “SYBR®”, or on capillaries (“LightCycler® Capillaries”). In someembodiments, the quantification of genetic material is determined byoptical absorbance and with real-time PCR. In some embodiments, thequantification of genetic material is determined by digital PCR. In someembodiments, the genes analyzed can be compared to a reference nucleicacid extract (DNA and RNA) corresponding to the expression (mRNA) andquantity (DNA) in order to compare expression levels of the targetnucleic acids.

(XIV) Antibody

An “antibody” is a polypeptide molecule that recognizes and binds to acomplementary target antigen. Antibodies typically have a molecularstructure shape that resembles a Y shape. Naturally-occurringantibodies, referred to as immunoglobulins, belong to one of theimmunoglobulin classes IgG, IgM, IgA, IgD, and IgE. Antibodies can alsobe produced synthetically. For example, recombinant antibodies, whichare monoclonal antibodies, can be synthesized using synthetic genes byrecovering the antibody genes from source cells, amplifying into anappropriate vector, and introducing the vector into a host to cause thehost to express the recombinant antibody. In general, recombinantantibodies can be cloned from any species of antibody-producing animalusing suitable oligonucleotide primers and/or hybridization probes.Recombinant techniques can be used to generate antibodies and antibodyfragments, including non-endogenous species.

Synthetic antibodies can be derived from non-immunoglobulin sources. Forexample, antibodies can be generated from nucleic acids (e.g.,aptamers), and from non-immunoglobulin protein scaffolds (such aspeptide aptamers) into which hypervariable loops are inserted to formantigen binding sites. Synthetic antibodies based on nucleic acids orpeptide structures can be smaller than immunoglobulin-derivedantibodies, leading to greater tissue penetration.

Antibodies can also include affimer proteins, which are affinityreagents that typically have a molecular weight of about 12-14 kDa.Affimer proteins generally bind to a target (e.g., a target protein)with both high affinity and specificity. Examples of such targetsinclude, but are not limited to, ubiquitin chains, immunoglobulins, andC-reactive protein. In some embodiments, affimer proteins are derivedfrom cysteine protease inhibitors, and include peptide loops and avariable N-terminal sequence that provides the binding site.

Antibodies can also include single domain antibodies (V_(H)H domains andVNAR domains), scFvs, and Fab fragments.

(XV) Affinity Group

An “affinity group” is a molecule or molecular moiety which has a highaffinity or preference for associating or binding with another specificor particular molecule or moiety. The association or binding withanother specific or particular molecule or moiety can be via anon-covalent interaction, such as hydrogen bonding, ionic forces, andvan der Waals interactions. An affinity group can, for example, bebiotin, which has a high affinity or preference to associate or bind tothe protein avidin or streptavidin. An affinity group, for example, canalso refer to avidin or streptavidin which has an affinity to biotin.Other examples of an affinity group and specific or particular moleculeor moiety to which it binds or associates with include, but are notlimited to, antibodies or antibody fragments and their respectiveantigens, such as digoxigenin and anti-digoxigenin antibodies, lectin,and carbohydrates (e.g., a sugar, a monosaccharide, a disaccharide, or apolysaccharide), and receptors and receptor ligands.

Any pair of affinity group and its specific or particular molecule ormoiety to which it binds or associates with can have their rolesreversed, for example, such that between a first molecule and a secondmolecule, in a first instance the first molecule is characterized as anaffinity group for the second molecule, and in a second instance thesecond molecule is characterized as an affinity group for the firstmolecule.

(XVI) Label, Detectable Label, and Optical Label

The terms “detectable label,” “optical label,” and “label” are usedinterchangeably herein to refer to a directly or indirectly detectablemoiety that is associated with (e.g., conjugated to) a molecule to bedetected, e.g., a capture probe or analyte. The detectable label can bedirectly detectable by itself (e.g., radioisotope labels or fluorescentlabels) or, in the case of an enzymatic label, can be indirectlydetectable, e.g., by catalyzing chemical alterations of a chemicalsubstrate compound or composition, which chemical substrate compound orcomposition is directly detectable. Detectable labels can be suitablefor small scale detection and/or suitable for high-throughput screening.As such, suitable detectable labels include, but are not limited to,radioisotopes, fluorophores, chemiluminescent compounds, bioluminescentcompounds, and dyes.

The detectable label can be qualitatively detected (e.g., optically orspectrally), or it can be quantified. Qualitative detection generallyincludes a detection method in which the existence or presence of thedetectable label is confirmed, whereas quantifiable detection generallyincludes a detection method having a quantifiable (e.g., numericallyreportable) value such as an intensity, duration, polarization, and/orother properties. In some embodiments, the detectable label is bound toa feature or to a capture probe associated with a feature. For example,detectably labeled features can include a fluorescent, a colorimetric,or a chemiluminescent label attached to a bead (see, for example,Rajeswari et al., J. Microbiol Methods 139:22-28, 2017, and Forcucci etal., J. Biomed Opt. 10:105010, 2015, the entire contents of each ofwhich are incorporated herein by reference).

In some embodiments, a plurality of detectable labels can be attached toa feature, capture probe, or composition to be detected. For example,detectable labels can be incorporated during nucleic acid polymerizationor amplification (e.g., Cy5®-labelled nucleotides, such asCy5^(g)-dCTP). Any suitable detectable label can be used. In someembodiments, the detectable label is a fluorophore. For example, thefluorophore can be from a group that includes: 7-AAD (7-AminoactinomycinD), Acridine Orange (+DNA), Acridine Orange (+RNA), Alexa Fluor® 350,Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546,Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, AlexaFluor® 633,Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700,Alexa Fluor® 750, Allophycocyanin (APC), AMCA / AMCA-X,7-Aminoactinomycin D (7-AAD), 7- Amino-4-methylcoumarin,6-Aminoquinoline, Aniline Blue, ANS, APC-Cy7, ATTO-TAG™ CBQCA, ATTO-TAG™FQ, Auramine O-Feulgen, BCECF (high pH), BFP (Blue Fluorescent Protein),BFP / GFP FRET, BOBO™-1/ BO-PRO™-1, BOBO™-3/ BO-PRO™-3, BODIPY® FL,BODIPY® TMR, BODIPY® TR-X, BODIPY® 530/550, BODIPY® 558/568, BODIPY®564/570, BODIPY® 581/591, BODIPY® 630/650-X, BODIPY® 650-665-X, BTC,Calcein, Calcein Blue, Calcium Crimson™, Calcium Green-1™, CalciumOrange™, Calcofluor® White, 5-Carboxyfluoroscein (5-FAM), 5-Carboxynaphthofluoroscein, 6-Carboxyrhodamine 6G,5-Carboxytetramethylrhodamine (5-TAMRA), Carboxy-X-rhodamine (5-ROX),Cascade Blue®, Cascade Yellow™, CCF2 (GeneBLAzer™), CFP (CyanFluorescent Protein), CFP / YFP FRET, Chromomycin A3, C1-NERF (low pH),CPM, 6-CR 6G, CTC Formazan, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy7®,Cychrome (PE-Cy5), Dansylamine, Dansyl cadaverine, Dansylchloride, DAPI,Dapoxyl, DCFH, DHR, DiA (4-Di-16-ASP), DiD (DilC18(5)), DIDS, Dil(DilC18(3)), DiO (DiOC18(3)), DiR (DilC18(7)), Di-4 ANEPPS, Di-8 ANEPPS,DM-NERF (4.5-6.5 pH), DsRed (Red Fluorescent Protein), EBFP, ECFP, EGFP,ELF® -97 alcohol, Eosin, Erythrosin, Ethidium bromide, Ethidiumhomodimer-1 (EthD-1), Europium (III) Chloride, 5-FAM(5-Carboxyfluorescein), Fast Blue, Fluorescein-dT phosphoramidite, FITC,Fluo-3, Fluo-4, FluorX®, Fluoro-Gold™ (high pH), Fluoro-Gold™ (low pH),Fluoro-Jade, FM® 1-43, Fura-2 (high calcium), Fura-2 / BCECF, Fura Red™(high calcium), Fura Red™ / Fluo-3, GeneBLAzer™ (CCF2), GFP Red Shifted(rsGFP), GFP Wild Type, GFP / BFP FRET, GFP / DsRed FRET, Hoechst 33342& 33258, 7-Hydroxy-4-methylcoumarin (pH 9), 1,5 IAEDANS, Indo-1 (highcalcium), Indo-1 (low calcium), Indodicarbocyanine, Indotricarbocyanine,JC-1, 6-JOE, JOJO™-1/ JO-PRO™-1, LDS 751 (+DNA), LDS 751 (+RNA), LOLO™-1/ LO-PRO™-1, Lucifer Yellow, LysoSensor™ Blue (pH 5), LysoSensor™ Green(pH 5), LysoSensor™ Yellow/Blue (pH 4.2), LysoTracker® Green,LysoTracker® Red, LysoTracker® Yellow, Mag-Fura-2, Mag-Indo-1, MagnesiumGreen™, Marina Blue®, 4-Methylumbelliferone, Mithramycin, MitoTracker®Green, MitoTracker® Orange, MitoTracker® Red, NBD (amine), Nile Red,Oregon Green® 488, Oregon Green® 500, Oregon Green® 514, Pacific Blue,PBF1, PE (R-phycoerythrin), PE-Cy5, PE-Cy7, PE-Texas Red, PerCP(Peridinin chlorphyll protein), PerCP-Cy5.5 (TruRed), PharRed (APC-Cy7),C-phycocyanin, R-phycocyanin, R-phycoerythrin (PE), PI (PropidiumIodide), PKH26, PKH67, POPO™-1 / PO-PRO™-1, POPO™-3 / PO-PRO™-3,Propidium Iodide (PI), PyMPO, Pyrene, Pyronin Y, Quantam Red (PE-Cy5),Quinacrine Mustard, R670 (PE-Cy5), Red 613 (PE-Texas Red), RedFluorescent Protein (DsRed), Resorufin, RH 414, Rhod-2, Rhodamine B,Rhodamine Green™, Rhodamine Red™, Rhodamine Phalloidin, Rhodamine 110,Rhodamine 123, 5-ROX (carboxy-X-rhodamine), S65A, S65C, S65L, S65T,SBFI, SITS, SNAFL®-1 (high pH), SNAFL®-2, SNARF®-1 (high pH), SNARF®-1(low pH), Sodium Green™, SpectrumAqua®, SpectrumGreen® #1,SpectrumGreen® #2, SpectrumOrange®, SpectrumRed®, SYTO® 11, SYTO® 13,SYTO® 17, SYTO® 45, SYTOX® Blue, SYTOX® Green, SYTOX® Orange, 5-TAMRA(5-Carboxytetramethylrhodamine), Tetramethylrhodamine (TRITC), TexasRed® / Texas Red®-X, Texas Red®-X (NHS Ester), Thiadicarbocyanine,Thiazole Orange, TOTO®-1 / TO-PRO®-1, TOTO®-3 / TO-PRO®-3, TO-PRO®-5,Tri-color (PE-Cy5), TRITC (Tetramethylrhodamine), TruRed (PerCP-Cy5.5),WW 781, X-Rhodamine (XRITC), Y66F, Y66H, Y66W, YFP (Yellow FluorescentProtein), YOYO®-1 / YO-PRO®-1, YOYO®-3 / YO-PRO®-3, 6-FAM (Fluorescein),6-FAM (NHS Ester), 6-FAM (Azide), HEX, TAMRA (NHS Ester), Yakima Yellow,MAX, TET, TEX615, ATTO 488, ATTO 532, ATTO 550, ATTO 565, ATTO Rho101,ATTO 590, ATTO 633, ATTO 647N, TYE 563, TYE 665, TYE 705, 5′ IRDye® 700,5′ IRDye® 800, 5′ IRDye® 800CW (NHS Ester), WellRED D4 Dye, WellRED D3Dye, WellRED D2 Dye, Lightcycler® 640 (NHS Ester), and Dy 750 (NHSEster).

As mentioned above, in some embodiments, a detectable label is orincludes a luminescent or chemiluminescent moiety. Commonluminescent/chemiluminescent moieties include, but are not limited to,peroxidases such as horseradish peroxidase (HRP), soybean peroxidase(SP), alkaline phosphatase, and luciferase. These protein moieties cancatalyze chemiluminescent reactions given the appropriate chemicalsubstrates (e.g., an oxidizing reagent plus a chemiluminescentcompound). A number of compound families are known to providechemiluminescence under a variety of conditions. Non-limiting examplesof chemiluminescent compound families include2,3-dihydro-1,4-phthalazinedione luminol, 5-amino-6,7,8-trimethoxy- andthe dimethylamino[ca]benz analog. These compounds can luminesce in thepresence of alkaline hydrogen peroxide or calcium hypochlorite and base.Other examples of chemiluminescent compound families include, e.g.,2,4,5-triphenylimidazoles, para-dimethylamino and - methoxysubstituents, oxalates such as oxalyl active esters, p-nitrophenyl,N-alkyl acridinum esters, luciferins, lucigenins, or acridinium esters.

(XVII) Template Switching Oligonucleotide

A “template switching oligonucleotide” is an oligonucleotide thathybridizes to untemplated nucleotides added by a reverse transcriptase(e.g., enzyme with terminal transferase activity) during reversetranscription. In some embodiments, a template switching oligonucleotidehybridizes to untemplated poly(C) nucleotides added by a reversetranscriptase. In some embodiments, the template switchingoligonucleotide adds a common 5′ sequence to full-length cDNA that isused for cDNA amplification.

In some embodiments, the template switching oligonucleotide adds acommon sequence onto the 5′ end of the RNA being reverse transcribed.For example, a template switching oligonucleotide can hybridize tountemplated poly(C) nucleotides added onto the end of a cDNA moleculeand provide a template for the reverse transcriptase to continuereplication to the 5′ end of the template switching oligonucleotide,thereby generating full-length cDNA ready for further amplification. Insome embodiments, once a full-length cDNA molecule is generated, thetemplate switching oligonucleotide can serve as a primer in a cDNAamplification reaction.

In some embodiments, a template switching oligonucleotide is addedbefore, contemporaneously with, or after a reverse transcription, orother terminal transferase-based reaction. In some embodiments, atemplate switching oligonucleotide is included in the capture probe. Incertain embodiments, methods of sample analysis using template switchingoligonucleotides can involve the generation of nucleic acid productsfrom analytes of the tissue sample, followed by further processing ofthe nucleic acid products with the template switching oligonucleotide.

Template switching oligonucleotides can include a hybridization regionand a template region. The hybridization region can include any sequencecapable of hybridizing to the target. In some embodiments, thehybridization region can, e.g., include a series of G bases tocomplement the overhanging C bases at the 3′ end of a cDNA molecule. Theseries of G bases can include 1 G base, 2 G bases, 3 G bases, 4 G bases,5 G bases, or more than 5 G bases. The template sequence can include anysequence to be incorporated into the cDNA. In other embodiments, thehybridization region can include at least one base in addition to atleast one G base. In other embodiments, the hybridization can includebases that are not a G base. In some embodiments, the template regionincludes at least 1 (e.g., at least 2, 3, 4, 5 or more) tag sequencesand/or functional sequences. In some embodiments, the template regionand hybridization region are separated by a spacer.

In some embodiments, the template regions include a barcode sequence.The barcode sequence can act as a spatial barcode and/or as a uniquemolecular identifier. Template switching oligonucleotides can includedeoxyribonucleic acids; ribonucleic acids; modified nucleic acidsincluding 2-aminopurine, 2,6-diaminopurine (2-amino-dA), inverted dT,5-methyl dC, 2′-deoxyInosine, Super T (5-hydroxybutynl-2′-deoxyuridine),Super G (8-aza-7-deazaguanosine), locked nucleic acids (LNAs), unlockednucleic acids (UNAs, e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC,2′ fluoro bases (e.g., Fluoro C, Fluoro U, Fluoro A, and Fluoro G), orany combination of the foregoing.

In some embodiments, the length of a template switching oligonucleotidecan be at least about 1, 2, 10, 20, 50, 75, 100, 150, 200, or 250nucleotides or longer. In some embodiments, the length of a templateswitching oligonucleotide can be at most about 2, 10, 20, 50, 100, 150,200, or 250 nucleotides or longer.

(XVIII) Splint Oligonucleotide

A “splint oligonucleotide” is an oligonucleotide that, when hybridizedto other polynucleotides, acts as a “splint” to position thepolynucleotides next to one another so that they can be ligatedtogether. In some embodiments, the splint oligonucleotide is DNA or RNA.The splint oligonucleotide can include a nucleotide sequence that ispartially complimentary to nucleotide sequences from two or moredifferent oligonucleotides. In some embodiments, the splintoligonucleotide assists in ligating a “donor” oligonucleotide and an“acceptor” oligonucleotide. In general, an RNA ligase, a DNA ligase, oranother other variety of ligase is used to ligate two nucleotidesequences together

In some embodiments, the splint oligonucleotide is between 10 and 50oligonucleotides in length, e.g., between 10 and 45, 10 and 40, 10 and35, 10 and 30, 10 and 25, or 10 and 20 oligonucleotides in length. Insome embodiments, the splint oligonucleotide is between 15 and 50, 15and 45, 15 and 40, 15 and 35, 15 and 30, 15 and 30, or 15 and 25nucleotides in length.

(C) Analytes

The apparatus, systems, methods, and compositions described in thisdisclosure can be used to detect and analyze a wide variety of differentanalytes. For the purpose of this disclosure, an “analyte” can includeany biological substance, structure, moiety, or component to beanalyzed. The term “target” can similarly refer to an analyte ofinterest.

Analytes can be broadly classified into one of two groups: nucleic acidanalytes, and non-nucleic acid analytes. Examples of non-nucleic acidanalytes include, but are not limited to, lipids, carbohydrates,peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins,phosphoproteins, specific phosphorylated or acetylated variants ofproteins, amidation variants of proteins, hydroxylation variants ofproteins, methylation variants of proteins, ubiquitylation variants ofproteins, sulfation variants of proteins, viral coat proteins,extracellular and intracellular proteins, antibodies, and antigenbinding fragments. In some embodiments, the analyte can be an organelle(e.g., nuclei or mitochondria).

Cell surface features corresponding to analytes can include, but are notlimited to, a receptor, an antigen, a surface protein, a transmembraneprotein, a cluster of differentiation protein, a protein channel, aprotein pump, a carrier protein, a phospholipid, a glycoprotein, aglycolipid, a cell-cell interaction protein complex, anantigen-presenting complex, a major histocompatibility complex, anengineered T-cell receptor, a T-cell receptor, a B-cell receptor, achimeric antigen receptor, an extracellular matrix protein, aposttranslational modification (e.g., phosphorylation, glycosylation,ubiquitination, nitrosylation, methylation, acetylation or lipidation)state of a cell surface protein, a gap junction, and an adherensjunction.

Analytes can be derived from a specific type of cell and/or a specificsub-cellular region. For example, analytes can be derived from cytosol,from cell nuclei, from mitochondria, from microsomes, and moregenerally, from any other compartment, organelle, or portion of a cell.Permeabilizing agents that specifically target certain cell compartmentsand organelles can be used to selectively release analytes from cellsfor analysis.

Examples of nucleic acid analytes include DNA analytes such as genomicDNA, methylated DNA, specific methylated DNA sequences, fragmented DNA,mitochondrial DNA, in situ synthesized PCR products, and RNA/DNAhybrids.

Examples of nucleic acid analytes also include RNA analytes such asvarious types of coding and non-coding RNA. Examples of the differenttypes of RNA analytes include messenger RNA (mRNA), ribosomal RNA(rRNA), transfer RNA (tRNA), microRNA (miRNA), and viral RNA. The RNAcan be a transcript (e.g., present in a tissue section). The RNA can besmall (e.g., less than 200 nucleic acid bases in length) or large (e.g.,RNA greater than 200 nucleic acid bases in length). Small RNAs mainlyinclude 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA),microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA(snoRNAs), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA),and small rDNA-derived RNA (srRNA). The RNA can be doublestranded RNA orsingle-stranded RNA. The RNA can be circular RNA. The RNA can be abacterial rRNA (e.g., 16s rRNA or 23s rRNA).

Additional examples of analytes include mRNA and cell surface features(e.g., using the labelling agents described herein), mRNA andintracellular proteins (e.g., transcription factors), mRNA and cellmethylation status, mRNA and accessible chromatin (e.g., ATAC-seq,DNase-seq, and/or MNase-seq), mRNA and metabolites (e.g., using thelabelling agents described herein), a barcoded labelling agent (e.g.,the oligonucleotide tagged antibodies described herein) and a V(D)Jsequence of an immune cell receptor (e.g., T-cell receptor), mRNA and aperturbation agent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc fingernuclease, and/or antisense oligonucleotide as described herein). In someembodiments, a perturbation agent can be a small molecule, an antibody,a drug, an aptamer, a miRNA, a physical environmental (e.g., temperaturechange), or any other known perturbation agents.

Analytes can include a nucleic acid molecule with a nucleic acidsequence encoding at least a portion of a V(D)J sequence of an immunecell receptor (e.g., a TCR or BCR). In some embodiments, the nucleicacid molecule is cDNA first generated from reverse transcription of thecorresponding mRNA, using a poly(T) containing primer. The generatedcDNA can then be barcoded using a capture probe, featuring a barcodesequence (and optionally, a UMI sequence) that hybridizes with at leasta portion of the generated cDNA. In some embodiments, a templateswitching oligonucleotide hybridizes to a poly(C) tail added to a 3′endof the cDNA by a reverse transcriptase enzyme. The original mRNAtemplate and template switching oligonucleotide can then be denaturedfrom the cDNA and the barcoded capture probe can then hybridize with thecDNA and a complement of the cDNA generated. Additional methods andcompositions suitable for barcoding cDNA generated from mRNA transcriptsincluding those encoding V(D)J regions of an immune cell receptor and/orbarcoding methods and composition including a template switcholigonucleotide are described in PCT Pat. Application PCT/US2017/057269,filed Oct. 18, 2017, and U.S. Pat. Application Serial No. 15/825,740,filed Nov. 29, 2017, both of which are incorporated herein by referencein their entireties. V(D)J analysis can also be completed with the useof one or more labelling agents that bind to particular surface featuresof immune cells and associated with barcode sequences. The one or morelabelling agents can include an MHC or MHC multimer.

As described above, the analyte can include a nucleic acid capable offunctioning as a component of a gene editing reaction, such as, forexample, clustered regularly interspaced short palindromic repeats(CRISPR)-based gene editing. Accordingly, the capture probe can includea nucleic acid sequence that is complementary to the analyte (e.g., asequence that can hybridize to the CRISPR RNA (crRNA), single guide RNA(sgRNA), or an adapter sequence engineered into a crRNA or sgRNA).

In certain embodiments, an analyte can be extracted from a live cell.Processing conditions can be adjusted to ensure that a biological sampleremains live during analysis, and analytes are extracted from (orreleased from) live cells of the sample. Live cell-derived analytes canbe obtained only once from the sample, or can be obtained at intervalsfrom a sample that continues to remain in viable condition.

In general, the systems, apparatus, methods, and compositions can beused to analyze any number of analytes. For example, the number ofanalytes that are analyzed can be at least about 2, at least about 3, atleast about 4, at least about 5, at least about 6, at least about 7, atleast about 8, at least about 9, at least about 10, at least about 11,at least about 12, at least about 13, at least about 14, at least about15, at least about 20, at least about 25, at least about 30, at leastabout 40, at least about 50, at least about 100, at least about 1,000,at least about 10,000, at least about 100,000 or more different analytespresent in a region of the sample or within an individual feature of thesubstrate. Methods for performing multiplexed assays to analyze two ormore different analytes will be discussed in a subsequent section ofthis disclosure.

(D) Biological Samples (I) Types of Biological Samples

A “biological sample” is obtained from the subject for analysis usingany of a variety of techniques including, but not limited to, biopsy,surgery, and laser capture microscopy (LCM), and generally includescells and/or other biological material from the subject. In addition tothe subjects described above, a biological sample can be obtained fromnon-mammalian organisms (e.g., a plants, an insect, an arachnid, anematode (e.g., Caenorhabditis elegans), a fungi, an amphibian, or afish (e.g., zebrafish)). A biological sample can be obtained from aprokaryote such as a bacterium, e.g., Escherichia coli, Staphylococci orMycoplasma pneumoniae; an archaea; a virus such as Hepatitis C virus orhuman immunodeficiency virus; or a viroid. A biological sample can beobtained from a eukaryote, such as a patient derived organoid (PDO) orpatient derived xenograft (PDX). The biological sample can includeorganoids, a miniaturized and simplified version of an organ produced invitro in three dimensions that shows realistic micro-anatomy. Organoidscan be generated from one or more cells from a tissue, embryonic stemcells, and/or induced pluripotent stem cells, which can self-organize inthree-dimensional culture owing to their self-renewal anddifferentiation capacities. In some embodiments, an organoid is acerebral organoid, an intestinal organoid, a stomach organoid, a lingualorganoid, a thyroid organoid, a thymic organoid, a testicular organoid,a hepatic organoid, a pancreatic organoid, an epithelial organoid, alung organoid, a kidney organoid, a gastruloid, a cardiac organoid, or aretinal organoid. Subjects from which biological samples can be obtainedcan be healthy or asymptomatic individuals, individuals that have or aresuspected of having a disease (e.g., cancer) or a pre-disposition to adisease, and/or individuals that are in need of therapy or suspected ofneeding therapy.

Biological samples can be derived from a homogeneous culture orpopulation of the subjects or organisms mentioned herein oralternatively from a collection of several different organisms, forexample, in a community or ecosystem.

Biological samples can include one or more diseased cells. A diseasedcell can have altered metabolic properties, gene expression, proteinexpression, and/or morphologic features. Examples of diseases includeinflammatory disorders, metabolic disorders, nervous system disorders,and cancer. Cancer cells can be derived from solid tumors, hematologicalmalignancies, cell lines, or obtained as circulating tumor cells.

Biological samples can also include fetal cells. For example, aprocedure such as amniocentesis can be performed to obtain a fetal cellsample from maternal circulation. Sequencing of fetal cells can be usedto identify any of a number of genetic disorders, including, e.g.,aneuploidy such as Down’s syndrome, Edwards syndrome, and Patausyndrome. Further, cell surface features of fetal cells can be used toidentify any of a number of disorders or diseases.

Biological samples can also include immune cells. Sequence analysis ofthe immune repertoire of such cells, including genomic, proteomic, andcell surface features, can provide a wealth of information to facilitatean understanding the status and function of the immune system. By way ofexample, determining the status (e.g., negative or positive) of minimalresidue disease (MRD) in a multiple myeloma (MM) patient followingautologous stem cell transplantation is considered a predictor of MRD inthe MM patient (see, e.g., U.S. Pat. Application Publication No.2018/0156784, the entire contents of which are incorporated herein byreference).

Examples of immune cells in a biological sample include, but are notlimited to, B cells, T cells (e.g., cytotoxic T cells, natural killer Tcells, regulatory T cells, and T helper cells), natural killer cells,cytokine induced killer (CIK) cells, myeloid cells, such as granulocytes(basophil granulocytes, eosinophil granulocytes, neutrophilgranulocytes/hypersegmented neutrophils), monocytes/macrophages, mastcells, thrombocytes/megakaryocytes, and dendritic cells.

The biological sample can include any number of macromolecules, forexample, cellular macromolecules and organelles (e.g., mitochondria andnuclei). The biological sample can be a nucleic acid sample and/orprotein sample. The biological sample can be a carbohydrate sample or alipid sample. The biological sample can be obtained as a tissue sample,such as a tissue section, biopsy, a core biopsy, needle aspirate, orfine needle aspirate. The sample can be a fluid sample, such as a bloodsample, urine sample, or saliva sample. The sample can be a skin sample,a colon sample, a cheek swab, a histology sample, a histopathologysample, a plasma or serum sample, a tumor sample, living cells, culturedcells, a clinical sample such as, for example, whole blood orblood-derived products, blood cells, or cultured tissues or cells,including cell suspensions.

Cell-free biological samples can include extracellular polynucleotides.Extracellular polynucleotides can be isolated from a bodily sample,e.g., blood, plasma, serum, urine, saliva, mucosal excretions, sputum,stool, and tears.

As discussed above, a biological sample can include a single analyte ofinterest, or more than one analyte of interest. Methods for performingmultiplexed assays to analyze two or more different analytes in a singlebiological sample is discussed in a subsequent section of thisdisclosure.

(II) Preparation of Biological Samples

A variety of steps can be performed to prepare a biological sample foranalysis. Except where indicated otherwise, the preparative stepsdescribed below can generally be combined in any manner to appropriatelyprepare a particular sample for analysis.

Tissue Sectioning

A biological sample can be harvested from a subject (e.g., via surgicalbiopsy, whole subject sectioning), grown in vitro on a growth substrateor culture dish as a population of cells, or prepared as a tissue sliceor tissue section. Grown samples may be sufficiently thin for analysiswithout further processing steps. Alternatively, grown samples, andsamples obtained via biopsy or sectioning, can be prepared as thintissue sections using a mechanical cutting apparatus such as a vibratingblade microtome. As another alternative, in some embodiments, a thintissue section can be prepared by applying a touch imprint of abiological sample to a suitable substrate material.

The thickness of the tissue section can be a fraction of (e.g., lessthan 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1) the maximumcross-sectional dimension of a cell. However, tissue sections having athickness that is larger than the maximum cross-section cell dimensioncan also be used. For example, cryostat sections can be used, which canbe, e.g., 10-20 micrometers thick.

More generally, the thickness of a tissue section typically depends onthe method used to prepare the section and the physical characteristicsof the tissue, and therefore sections having a wide variety of differentthicknesses can be prepared and used. For example, the thickness of thetissue section can be at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1.0, 1.5,2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 20, 30, 40, or 50micrometers. Thicker sections can also be used if desired or convenient,e.g., at least 70, 80, 90, or 100 micrometers or more. Typically, thethickness of a tissue section is between 1-100 micrometers, 1-50micrometers, 1-30 micrometers, 1-25 micrometers, 1-20 micrometers, 1-15micrometers, 1-10 micrometers, 2-8 micrometers, 3-7 micrometers, or 4-6micrometers, but as mentioned above, sections with thicknesses larger orsmaller than these ranges can also be analysed.

Multiple sections can also be obtained from a single biological sample.For example, multiple tissue sections can be obtained from a surgicalbiopsy sample by performing serial sectioning of the biopsy sample usinga sectioning blade. Spatial information among the serial sections can bepreserved in this manner, and the sections can be analysed successivelyto obtain three-dimensional information about the biological sample.

Freezing

In some embodiments, the biological sample (e.g., a tissue section asdescribed above) can be prepared by deep freezing at a temperaturesuitable to maintain or preserve the integrity (e.g., the physicalcharacteristics) of the tissue structure. Such a temperature can be,e.g., less than -20° C., or less than -25° C., -30° C., -40° C., -50°C., -60° C., -70° C., -80° C. -90° C., - 100° C., -110° C., -120° C.,-130° C., -140° C., -150° C., -160° C., -170° C., -180° C., -190° C., or-200° C. The frozen tissue sample can be sectioned, e.g., thinly sliced,onto a substrate surface using any number of suitable methods. Forexample, a tissue sample can be prepared using a chilled microtome(e.g., a cryostat) set at a temperature suitable to maintain both thestructural integrity of the tissue sample and the chemical properties ofthe nucleic acids in the sample. Such a temperature can be, e.g., lessthan -15° C., less than -20° C., or less than -25° C. A sample can besnap frozen in isopentane and liquid nitrogen. Frozen samples can bestored in a sealed container prior to embedding.

Formalin Fixation and Paraffin Embedding

In some embodiments, the biological sample can be prepared usingformalin-fixation and paraffin-embedding (FFPE), which are establishedmethods. In some embodiments, cell suspensions and other non-tissuesamples can be prepared using formalin-fixation and paraffin-embedding.Following fixation of the sample and embedding in a paraffin or resinblock, the sample can be sectioned as described above. Prior toanalysis, the paraffin-embedding material can be removed from the tissuesection (e.g., deparaffinization) by incubating the tissue section in anappropriate solvent (e.g., xylene) followed by a rinse (e.g., 99.5%ethanol for 2 minutes, 96% ethanol for 2 minutes, and 70% ethanol for 2minutes).

Fixation

As an alternative to formalin fixation described above, a biologicalsample can be fixed in any of a variety of other fixatives to preservethe biological structure of the sample prior to analysis. For example, asample can be fixed via immersion in ethanol, methanol, acetone,formaldehyde (e.g., 2% formaldehyde), paraformaldehyde-Triton,glutaraldehyde, or combinations thereof.

In some embodiments, acetone fixation is used with fresh frozen samples,which can include, but are not limited to, cortex tissue, mouseolfactory bulb, human brain tumor, human post-mortem brain, and breastcancer samples. In some embodiments, a compatible fixation method ischosen and/or optimized based on a desired workflow. For example,formaldehyde fixation may be chosen as compatible for workflows usingIHC/IF protocols for protein visualization. As another example, methanolfixation may be chosen for workflows emphasizing RNA/DNA libraryquality. Acetone fixation may be chosen in some applications topermeabilize the tissue. When acetone fixation is performed,pre-permeabilization steps (described below) may not be performed.Alternatively, acetone fixation can be performed in conjunction withpermeabilization steps.

Embedding

As an alternative to paraffin embedding described above, a biologicalsample can be embedded in any of a variety of other embedding materialsto provide a substrate to the sample prior to sectioning and otherhandling steps. In general, the embedding material is removed prior toanalysis of tissue sections obtained from the sample. Suitable embeddingmaterials include, but are not limited to, waxes, resins (e.g.,methacrylate resins), epoxies, and agar.

Staining

To facilitate visualization, biological samples can be stained using awide variety of stains and staining techniques. In some embodiments, asample can be stained using any number of biological stains, includingbut not limited to, acridine orange, Bismarck brown, carmine, coomassieblue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine,hematoxylin, Hoechst stains, iodine, methyl green, methylene blue,neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide,rhodamine, or safranin.

The sample can be stained using known staining techniques, includingCan-Grunwald, Giemsa, hematoxylin and eosin (H&E), Jenner’s, Leishman,Masson’s trichrome, Papanicolaou, Romanowsky, silver, Sudan, Wright’s,and/or Periodic Acid Schiff (PAS) staining techniques. PAS staining istypically performed after formalin or acetone fixation.

In some embodiments, the biological sample can be stained using adetectable label (e.g., radioisotopes, fluorophores, chemiluminescentcompounds, bioluminescent compounds, and dyes) as described elsewhereherein. In some embodiments, a biological sample is stained using onlyone type of stain or one technique. In some embodiments, stainingincludes biological staining techniques such as H&E staining. In someembodiments, staining includes identifying analytes usingfluorescently-conjugated antibodies. In some embodiments, a biologicalsample is stained using two or more different types of stains, or two ormore different staining techniques. For example, a biological sample canbe prepared by staining and imaging using one technique (e.g., H&Estaining and brightfield imaging), followed by staining and imagingusing another technique (e.g., IHC/IF staining and fluorescencemicroscopy) for the same biological sample.

In some embodiments, biological samples can be destained. Methods ofdestaining or discoloring a biological sample are known in the art, andgenerally depend on the nature of the stain(s) applied to the sample.For example, H&E staining can be destined by washing the sample in HCl.In some embodiments, destaining can include 1, 2, 3, or more washes inHCl. In some embodiments, destaining can include adding HCl to adownstream solution (e.g., permeabilization solution). As anotherexample, in some embodiments, one or more immunofluorescence stains areapplied to the sample via antibody coupling. Such stains can be removedusing techniques such as cleavage of disulfide linkages via treatmentwith a reducing agent and detergent washing, chaotropic salt treatment,treatment with antigen retrieval solution, and treatment with an acidicglycine buffer. Methods for multiplexed staining and destaining aredescribed, for example, in Bolognesi et al., J. Histochem. Cytochem.2017; 65(8): 431-444, Lin et al., Nat Commun. 2015; 6:8390, Pirici etal., J. Histochem. Cytochem. 2009; 57:567-75, and Glass et al., J.Histochem. Cytochem. 2009; 57:899-905, the entire contents of each ofwhich are incorporated herein by reference.

Hydrogel Embedding

In some embodiments, hydrogel formation occurs within a biologicalsample. In some embodiments, a biological sample (e.g., tissue section)is embedded in a hydrogel. In some embodiments, hydrogel subunits areinfused into the biological sample, and polymerization of the hydrogelis initiated by an external or internal stimulus. A “hydrogel” asdescribed herein can include a cross-linked 3D network of hydrophilicpolymer chains. A “hydrogel subunit” can be a hydrophilic monomer, amolecular precursor, or a polymer that can be polymerized (e.g.,cross-linked) to form a three-dimensional (3D) hydrogel network.

A hydrogel can swell in the presence of water. In some embodiments, ahydrogel comprises a natural material. In some embodiments, a hydrogelincludes a synthetic material. In some embodiments, a hydrogel includesa hybrid material, e.g., the hydrogel material comprises elements ofboth synthetic and natural polymers. Any of the materials used inhydrogels or hydrogels comprising a polypeptide-based material describedherein can be used. Embedding the sample in this manner typicallyinvolves contacting the biological sample with a hydrogel such that thebiological sample becomes surrounded by the hydrogel. For example, thesample can be embedded by contacting the sample with a suitable polymermaterial, and activating the polymer material to form a hydrogel. Insome embodiments, the hydrogel is formed such that the hydrogel isinternalized within the biological sample.

In some embodiments, the biological sample is immobilized in thehydrogel via cross-linking of the polymer material that forms thehydrogel. Cross-linking can be performed chemically and/orphotochemically, or alternatively by any other hydrogel-formation methodknown in the art. For example, the biological sample can be immobilizedin the hydrogel by polyacrylamide crosslinking. Further, analytes of abiological sample can be immobilized in a hydrogel by crosslinking(e.g., polyacrylamide crosslinking).

The composition and application of the hydrogel to a biological sampletypically depends on the nature and preparation of the biological sample(e.g., sectioned, non-sectioned, fresh-frozen tissue, type of fixation).A hydrogel can be any appropriate hydrogel where upon formation of thehydrogel on the biological sample the biological sample becomes anchoredto or embedded in the hydrogel. Non-limiting examples of hydrogels aredescribed herein or are known in the art. As one example, where thebiological sample is a tissue section, the hydrogel can include amonomer solution and an ammonium persulfate (APS)initiator/tetramethylethylenediamine (TEMED) accelerator solution. Asanother example, where the biological sample consists of cells (e.g.,cultured cells or cells disassociated from a tissue sample), the cellscan be incubated with the monomer solution and APS/TEMED solutions. Forcells, hydrogel are formed in compartments, including but not limited todevices used to culture, maintain, or transport the cells. For example,hydrogels can be formed with monomer solution plus APS/TEMED added tothe compartment to a depth ranging from about 0.1 µm to about 5 mm.

In some embodiments, a hydrogel includes a linker that allows anchoringof the biological sample to the hydrogel. In some embodiments, ahydrogel includes linkers that allow anchoring of biological analytes tothe hydrogel. In such cases, the linker can be added to the hydrogelbefore, contemporaneously with, or after hydrogel formation.Non-limiting examples of linkers that anchor nucleic acids to thehydrogel can include 6-((Acryloyl)amino) hexanoic acid (Acryloyl-X SE)(available from ThermoFisher, Waltham, MA), Label-IT Amine (availablefrom MirusBio, Madison, WI) and Label X).

In some embodiments, functionalization chemistry can be used. In someembodiments, functionalization chemistry includes hydrogel-tissuechemistry (HTC). Any hydrogel-tissue backbone (e.g., synthetic ornative) suitable for HTC can be used for anchoring biologicalmacromolecules and modulating functionalization. Non-limiting examplesof methods using HTC backbone variants include CLARITY, PACT, ExM,SWITCH and ePACT. In some embodiments, hydrogel formation within abiological sample is permanent. For example, biological macromoleculescan permanently adhere to the hydrogel allowing multiple rounds ofinterrogation. In some embodiments, hydrogel formation within abiological sample is reversible.

In some embodiments, additional reagents are added to the hydrogelsubunits before, contemporaneously with, and/or after polymerization.For example, additional reagents can include but are not limited tooligonucleotides (e.g., capture probes), endonucleases to fragment DNA,fragmentation buffer for DNA, DNA polymerase enzymes, dNTPs used toamplify the nucleic acid and to attach the barcode to the amplifiedfragments. Other enzymes can be used, including without limitation, RNApolymerase, transposase, ligase, proteinase K, and DNAse. Additionalreagents can also include reverse transcriptase enzymes, includingenzymes with terminal transferase activity, primers, and switcholigonucleotides. In some embodiments, optical labels are added to thehydrogel subunits before, contemporaneously with, and/or afterpolymerization.

In some embodiments, HTC reagents are added to the hydrogel before,contemporaneously with, and/or after polymerization. In someembodiments, an analyte capture agent is added to the hydrogel before,contemporaneously with, and/or after polymerization. In someembodiments, a cell-penetrating agent is added to the hydrogel before,contemporaneously with, and/or after polymerization.

In some embodiments, a biological sample is embedded in a hydrogel tofacilitate sample transfer to another location (e.g., to an array). Forexample, archived biological samples (e.g., FFPE tissue sections) can betransferred from storage to a spatial array to perform spatial analysis.In some embodiments, a biological sample on a substrate can be coveredwith any of the prepolymer solutions described herein. In someembodiments, the prepolymer solution can be polymerized such that ahydrogel is formed on top of and/or around the biological sample.Hydrogel formation can occur in a manner sufficient to anchor (e.g.,embed) the biological sample to the hydrogel. After hydrogel formation,the biological sample is anchored to (e.g., embedded in) the hydrogelwherein separating the hydrogel from the substrate (e.g., glass slide)results in the biological sample separating from the substrate alongwith the hydrogel. The biological sample contained in the hydrogel canthen be contacted with a spatial array, and spatial analysis can beperformed on the biological sample.

Any variety of characteristics can determine the transfer conditionsrequired for a given biological sample. Non-limiting examples ofcharacteristics likely to impact transfer conditions include the sample(e.g., thickness, fixation, and cross-linking) and/or the analyte ofinterest (different conditions to preserve and/or transfer differentanalytes (e.g., DNA, RNA, and protein)).

In some embodiments, the hydrogel is removed after contacting thebiological sample with the spatial array. For example, methods describedherein can include an event-dependent (e.g., light or chemical)depolymerizing hydrogel, wherein upon application of the event (e.g.,external stimuli) the hydrogel depolymerizes. In one example, abiological sample can be anchored to a DTT-sensitive hydrogel, whereaddition of DTT can cause the hydrogel to depolymerize and release theanchored biological sample.

Hydrogels embedded within biological samples can be cleared using anysuitable method. For example, electrophoretic tissue clearing methodscan be used to remove biological macromolecules from thehydrogel-embedded sample. In some embodiments, a hydrogel-embeddedsample is stored in a medium before or after clearing of hydrogel (e.g.,a mounting medium, methylcellulose, or other semi-solid mediums).

In some embodiments, the hydrogel chemistry can be tuned to specificallybind (e.g., retain) particular species of analytes (e.g., RNA, DNA,protein, etc.). In some embodiments, a hydrogel includes a linker thatallows anchoring of the biological sample to the hydrogel. In someembodiments, a hydrogel includes linkers that allow anchoring ofbiological analytes to the hydrogel. In such cases, the linker can beadded to the hydrogel before, contemporaneously with, or after hydrogelformation. Non-limiting examples of linkers that anchor nucleic acids tothe hydrogel can include 6-((Acryloyl)amino) hexanoic acid (Acryloyl-XSE), Label-IT Amine and Label X. Non-limiting examples ofcharacteristics likely to impact transfer conditions include the sample(e.g., thickness, fixation, and cross-linking) and/or the analyte ofinterest (different conditions to preserve and/or transfer differentanalytes (e.g., DNA, RNA, and protein)).

Additional methods and aspects of hydrogel embedding of biologicalsamples are described for example in Chen et al., Science347(6221):543-548, 2015, the entire contents of which are incorporatedherein by reference.

Biological Sample Transfer

In some embodiments, a biological sample immobilized on a substrate(e.g., a biological sample prepared using methanol fixation orformalin-fixation and paraffin-embedding (FFPE)) is transferred to aspatial array using a hydrogel. In some embodiments, a hydrogel isformed on top of a biological sample on a substrate (e.g., glass slide).For example, hydrogel formation can occur in a manner sufficient toanchor (e.g., embed) the biological sample to the hydrogel. Afterhydrogel formation, the biological sample is anchored to (e.g., embeddedin) the hydrogel wherein separating the hydrogel from the substrateresults in the biological sample separating from the substrate alongwith the hydrogel. The biological sample can then be contacted with aspatial array, thereby allowing spatial profiling of the biologicalsample. In some embodiments, the hydrogel is removed after contactingthe biological sample with the spatial array. For example, methodsdescribed herein can include an event-dependent (e.g., light orchemical) depolymerizing hydrogel, wherein upon application of the event(e.g., external stimuli) the hydrogel depolymerizes. In one example, abiological sample can be anchored to a DTT-sensitive hydrogel, whereaddition of DTT can cause the hydrogel to depolymerize and release theanchored biological sample. A hydrogel can be any appropriate hydrogelwhere upon formation of the hydrogel on the biological sample thebiological sample becomes anchored to or embedded in the hydrogel.Non-limiting examples of hydrogels are described herein or are known inthe art. In some embodiments, a hydrogel includes a linker that allowsanchoring of the biological sample to the hydrogel. In some embodiments,a hydrogel includes linkers that allow anchoring of biological analytesto the hydrogel. In such cases, the linker can be added to the hydrogelbefore, contemporaneously with, or after hydrogel formation.Non-limiting examples of linkers that anchor nucleic acids to thehydrogel can include 6-((Acryloyl)amino) hexanoic acid (Acryloyl-X SE)(available from ThermoFisher, Waltham, MA), Label-IT Amine (availablefrom MirusBio, Madison, WI) and Label X). Any variety of characteristicscan determine the transfer conditions required for a given biologicalsample. Non-limiting examples of characteristics likely to impacttransfer conditions include the sample (e.g., thickness, fixation, andcross-linking) and/or the analyte of interest (different conditions topreserve and/or transfer different analytes (e.g., DNA, RNA, andprotein)). In some embodiments, hydrogel formation can occur in a mannersufficient to anchor the analytes (e.g., embed) in the biological sampleto the hydrogel. In some embodiments, the hydrogel can be imploded(e.g., shrunk) with the anchored analytes (e.g., embedded in thehydrogel) present in the biological sample. In some embodiments, thehydrogel can be expanded (e.g., isometric expansion) with the anchoredanalytes (e.g., embedded in the hydrogel) present in the biologicalsample. In some embodiments, the hydrogel can be imploded (e.g., shrunk)and subsequently expanded with anchored analytes (e.g., embedded in thehydrogel) present in the biological sample.

Isometric Expansion

In some embodiments, a biological sample embedded in a hydrogel can beisometrically expanded. Isometric expansion methods that can be usedinclude hydration, a preparative step in expansion microscopy, asdescribed in Chen et al., Science 347(6221):543-548, 2015; Asano et al.Current Protocols. 2018, 80:1, doi:10.1002/cpcb.56 and Gao et al. BMCBiology. 2017, 15:50, doi:10.1186/s12915-017-0393-3, Wassie, A.T., etal, Expansion microscopy: principles and uses in biological research,Nature Methods, 16(1): 33-41 (2018), each of which is incorporated byreference in its entirety.

In general, the steps used to perform isometric expansion of thebiological sample can depend on the characteristics of the sample (e.g.,thickness of tissue section, fixation, cross-linking), and/or theanalyte of interest (e.g., different conditions to anchor RNA, DNA, andprotein to a gel).

Isometric expansion can be performed by anchoring one or more componentsof a biological sample to a gel, followed by gel formation, proteolysis,and swelling. Isometric expansion of the biological sample can occurprior to immobilization of the biological sample on a substrate, orafter the biological sample is immobilized to a substrate. In someembodiments, the isometrically expanded biological sample can be removedfrom the substrate prior to contacting the expanded biological samplewith a spatially barcoded array (e.g., spatially barcoded capture probeson a substrate).

In some embodiments, proteins in the biological sample are anchored to aswellable gel such as a polyelectrolyte gel. An antibody can be directedto the protein before, after, or in conjunction with being anchored tothe swellable gel. DNA and/or RNA in a biological sample can also beanchored to the swellable gel via a suitable linker. Examples of suchlinkers include, but are not limited to, 6-((Acryloyl)amino) hexanoicacid (Acryloyl-X SE) (available from ThermoFisher, Waltham, MA),Label-IT Amine (available from MirusBio, Madison, WI) and Label X(described for example in Chen et al., Nat. Methods 13:679-684, 2016,the entire contents of which are incorporated herein by reference).

Isometric expansion of the sample can increase the spatial resolution ofthe subsequent analysis of the sample. For example, isometric expansionof the biological sample can result in increased resolution in spatialprofiling (e.g., single-cell profiling). The increased resolution inspatial profiling can be determined by comparison of an isometricallyexpanded sample with a sample that has not been isometrically expanded.

In some embodiments, a biological sample is isometrically expanded to avolume at least 2x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x,2.9x, 3x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4x,4.1x, 4.2x, 4.3x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, or 4.9x its non-expandedvolume. In some embodiments, the sample is isometrically expanded to atleast 2x and less than 20x of its non-expanded volume.

In some embodiments, a biological sample embedded in a hydrogel isisometrically expanded to a volume at least 2x, 2.1x, 2.2x, 2.3x, 2.4x,2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x,3.7x, 3.8x, 3.9x, 4x, 4.1x, 4.2x, 4.3x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, or4.9x its non-expanded volume. In some embodiments, the biological sampleembedded in a hydrogel is isometrically expanded to at least 2x and lessthan 20x of its non-expanded volume.

Substrate Attachment

In some embodiments, the biological sample can be attached to asubstrate. Examples of substrates suitable for this purpose aredescribed in detail below. Attachment of the biological sample can beirreversible or reversible, depending upon the nature of the sample andsubsequent steps in the analytical method.

In certain embodiments, the sample can be attached to the substratereversibly by applying a suitable polymer coating to the substrate, andcontacting the sample to the polymer coating. The sample can then bedetached from the substrate using an organic solvent that at leastpartially dissolves the polymer coating. Hydrogels are examples ofpolymers that are suitable for this purpose.

More generally, in some embodiments, the substrate can be coated orfunctionalized with one or more substances to facilitate attachment ofthe sample to the substrate. Suitable substances that can be used tocoat or functionalize the substrate include, but are not limited to,lectins, poly-lysine, antibodies, and polysaccharides.

Unaggregated Cells

In some embodiments, the biological sample corresponds to cells (e.g.,derived from a cell culture or a tissue sample). In a cell sample with aplurality of cells, individual cells can be naturally unaggregated. Forexample, cells can be derived from a suspension of cells and/ordisassociated or disaggregated cells from a tissue or tissue section.

Alternatively, the cells in the sample may be aggregated, and may bedisaggregated into individual cells using, for example, enzymatic ormechanical techniques. Examples of enzymes used in enzymaticdisaggregation include, but are not limited to, dispase, collagenase,trypsin, or combinations thereof. Mechanical disaggregation can beperformed, for example, using a tissue homogenizer.

In some embodiments of unaggregated cells or disaggregated cells, thecells are distributed onto the substrate such that at least one celloccupies a distinct spatial feature on the substrate. The cells can beimmobilized on the substrate (e.g., to prevent lateral diffusion of thecells). In some embodiments, a cell immobilization agent can be used toimmobilize a non-aggregated or disaggregated sample on aspatially-barcoded array prior to analyte capture. A “cellimmobilization agent” can refer to an antibody, attached to a substrate,which can bind to a cell surface marker. In some embodiments, thedistribution of the plurality of cells on the substrate follows Poissonstatistics.

In some embodiments, cells from a plurality of cells are immobilized ona substrate. In some embodiments, the cells are immobilized to preventlateral diffusion, for example, by adding a hydrogel and/or by theapplication of an electric field.

Suspended and Adherent Cells

In some embodiments, the biological sample can be derived from a cellculture grown in vitro. Samples derived from a cell culture can includeone or more suspension cells which are anchorage-independent within thecell culture. Examples of such cells include, but are not limited to,cell lines derived from hematopoietic cells, and from the following celllines: Colo205, CCRF-CEM, HL-60, K562, MOLT-4, RPMI-8226, SR, HOP-92,NCI-H322M, and MALME-3M.

Samples derived from a cell culture can include one or more adherentcells which grow on the surface of the vessel that contains the culturemedium. Non-limiting examples of adherent cells include DU145 (prostatecancer) cells, H295R (adrenocortical cancer) cells, HeLa (cervicalcancer) cells, KBM-7 (chronic myelogenous leukemia) cells, LNCaP(prostate cancer) cells, MCF-7 (breast cancer) cells, MDA-MB-468 (breastcancer) cells, PC3 (prostate cancer) cells, SaOS-2 (bone cancer) cells,SH-SY5Y (neuroblastoma, cloned from a myeloma) cells, T-47D (breastcancer) cells, THP-1 (acute myeloid leukemia) cells, U87 (glioblastoma)cells, National Cancer Institute’s 60 cancer cell line panel (NCI60),vero (African green monkey Chlorocebus kidney epithelial cell line)cells, MC3T3 (embryonic calvarium) cells, GH3 (pituitary tumor) cells,PC12 (pheochromocytoma) cells, dog MDCK kidney epithelial cells, XenopusA6 kidney epithelial cells, zebrafish AB9 cells, and Sf9 insectepithelial cells.

Additional examples of adherent cells are shown in Table 1 andcatalogued, for example, in “A Catalog of in Vitro Cell Lines,Transplantable Animal and Human Tumors and Yeast,” The Division ofCancer Treatment and Diagnosis (DCTD), National Cancer Institute (2013),and in Abaan et al., “The exomes of the NCI-60 panel: a genomic resourcefor cancer biology and systems pharmacology,” Cancer Research73(14):4372-82, 2013, the entire contents of each of which areincorporated by reference herein.

TABLE 1 Examples of adherent cells Cell Line Species Organ of OriginDisease BT549 Human Breast Ductal Carcinoma HS 578T Human BreastCarcinoma MCF7 Human Breast Adenocarcinoma MDA-MB-231 Human BreastAdenocarcinoma MDA-MB-468 Human Breast Adenocarcinoma T-47D Human BreastDuctal Carcinoma SF268 Human CNS Anaplastic Astrocytoma SF295 Human CNSGlioblastoma-Multiforme SF539 Human CNS Glioblastoma SNB-19 Human CNSGlioblastoma SNB-75 Human CNS Astrocytoma U251 Human CNS GlioblastomaColo205 Human Colon Dukes’ type D, Colorectal adenocarcinoma HCC 2998Human Colon Carcinoma HCT-116 Human Colon Carcinoma HCT-15 Human ColonDukes’ type C, Colorectal adenocarcinoma HT29 Human Colon Colorectaladenocarcinoma KM12 Human Colon Adenocarcinoma, Grade III SW620 HumanColon Adenocarcinoma 786-O Human Kidney renal cell adenocarcinoma A498Human Kidney Adenocarcinoma ACHN Human Kidney renal cell adenocarcinomaCAKI Human Kidney clear cell carcinoma RXF 393 Human Kidney PoorlyDifferentiated Hypernephroma SN12C Human Kidney Carcinoma TK-10 HumanKidney Spindle Cell carcinoma UO-31 Human Kidney Carcinoma A549 HumanLung Adenocarcinoma EKVX Human Lung Adenocarcinoma HOP-62 Human LungAdenocarcinoma HOP-92 Human Lung Large Cell, Undifferentiated NCI-H226Human Lung squamous cell carcinoma; mesothelioma NCI-H23 Human Lungadenocarcinoma; non-small cell lung cancer NCI-H460 Human Lungcarcinoma; large cell lung cancer NCI-H522 Human Lung adenocarcinoma;non-small cell lung cancer LOX IMVI Human Melanoma Malignant Amelanoticmelanoma M14 Human Melanoma malignant melanoma MALME-3M Human Melanomamalignant melanoma MDA-MB-435 Human Melanoma Adenocarcinoma SK-MEL-2Human Melanoma malignant melanoma SK-MEL-28 Human Melanoma malignantmelanoma SK-MEL-5 Human Melanoma malignant melanoma UACC-257 HumanMelanoma malignant melanoma UACC-62 Human Melanoma malignant melanomaIGROV1 Human Ovary Cystoadenocarcinoma OVCAR-3 Human OvaryAdenocarcinoma OVCAR-4 Human Ovary Adenocarcinoma OVCAR-5 Human OvaryAdenocarcinoma OVCAR-8 Human Ovary Adenocarcinoma SK-OV-3 Human OvaryAdenocarcinoma NCI-ADR-RES Human Ovary Adenocarcinoma DU145 HumanProstate Carcinoma PC-3 Human Prostate grade IV, adenocarcinoma

In some embodiments, the adherent cells are cells that correspond to oneor more of the following cell lines: BT549, HS 578T, MCF7, MDA-MB-231,MDA-MB-468, T-47D, SF268, SF295, SF539, SNB-19, SNB-75, U251, Colo205,HCC 2998, HCT-116, HCT-15, HT29, KM12, SW620, 786-O, A498, ACHN, CAKI,RXF 393, SN12C, TK-10, UO-31, A549, EKVX, HOP-62, HOP-92, NCI-H226,NCI-H23, NCI-H460, NCI-H522, LOX IMVI, M14, MALME-3M, MDA-MB-435, SK-,EL-2, SK-MEL-28, SK-MEL-5, UACC-257, UACC-62, IGROV1, OVCAR-3, OVCAR-4,OVCAR-5, OVCAR-8, SK-OV-3, NCI-ADR- RES, DU145, PC-3, DU145, H295R,HeLa, KBM-7, LNCaP, MCF-7, MDA-MB-468, PC3, SaOS-2, SH-SY5Y, T-47D,THP-1, U87, vero, MC3T3, GH3, PC12, dog MDCK kidney epithelial, XenopusA6 kidney epithelial, zebrafish AB9, and Sf9 insect epithelial celllines.

Tissue Permeabilization

In some embodiments, a biological sample can be permeabilized tofacilitate transfer of analytes out of the sample, and/or to facilitatetransfer of species (such as capture probes) into the sample. If asample is not permeabilized sufficiently, the amount of analyte capturedfrom the sample may be too low to enable adequate analysis. Conversely,if the tissue sample is too permeable, the relative spatial relationshipof the analytes within the tissue sample can be lost. Hence, a balancebetween permeabilizing the tissue sample enough to obtain good signalintensity while still maintaining the spatial resolution of the analytedistribution in the sample is desirable.

In general, a biological sample can be permeabilized by exposing thesample to one or more permeabilizing agents. Suitable agents for thispurpose include, but are not limited to, organic solvents (e.g.,acetone, ethanol, and methanol), cross-linking agents (e.g.,paraformaldehyde), detergents (e.g., saponin, Triton X-100™, Tween-20™,or sodium dodecyl sulfate (SDS)), and enzymes (e.g., trypsin, proteases(e.g., proteinase K). In some embodiments, the detergent is an anionicdetergent (e.g., SDS or N-lauroylsarcosine sodium salt solution). Insome embodiments, the biological sample can be permeabilized using anyof the methods described herein (e.g., using any of the detergentsdescribed herein, e.g., SDS and/or N-lauroylsarcosine sodium saltsolution) before or after enzymatic treatment (e.g., treatment with anyof the enzymes described herein, e.g., trypin, proteases (e.g., pepsinand/or proteinase K)).

In some embodiments, a biological sample can be permeabilized byexposing the sample to greater than about 1.0 w/v % (e.g., greater thanabout 2.0 w/v %, greater than about 3.0 w/v %, greater than about 4.0w/v%, greater than about 5.0 w/v %, greater than about 6.0 w/v %,greater than about 7.0 w/v %, greater than about 8.0 w/v %, greater thanabout 9.0 w/v %, greater than about 10.0 w/v %, greater than about 11.0w/v %, greater than about 12.0 w/v %, or greater than about 13.0 w/v %)sodium dodecyl sulfate (SDS) and/or N-lauroylsarcosine orN-lauroylsarcosine sodium salt. In some embodiments, a biological samplecan be permeabilized by exposing the sample (e.g., for about 5 minutesto about 1 hour, about 5 minutes to about 40 minutes, about 5 minutes toabout 30 minutes, about 5 minutes to about 20 minutes, or about 5minutes to about 10 minutes) to about 1.0 w/v % to about 14.0 w/v %(e.g., about 2.0 w/v % to about 14.0 w/v %, about 2.0 w/v % to about12.0w/v %, about 2.0 w/v % to about 10.0 w/v %, about 4.0 w/v % to about14.0 w/v %, about 4.0w/v % to about 12.0 w/v %, about 4.0 w/v % to about10.0 w/v %, about 6.0 w/v % to about 14.0 w/v %, about 6.0 w/v % toabout 12.0 w/v %, about 6.0 w/v % to about 10.0 w/v %, about 8.0 w/v %to about 14.0 w/v %, about 8.0 w/v % to about 12.0 w/v %, about 8.0 w/v% to about 10.0 w/v %, about 10.0 w/v %to about 14.0 w/v %, about 10.0w/v % to about 12.0 w/v %, or about 12.0 w/v % to about 14.0 w/v %) SDSand/or N-lauroylsarcosine salt solution and/or proteinase K (e.g., at atemperature of about 4% to about 35° C., about 4° C. to about 25° C.,about 4° C. to about 20° C., about 4° C. to about 10° C., about 10° C.to about 25° C., about 10° C. to about 20° C., about 10° C. to about 15°C., about 35° C. to about 50° C., about 35° C. to about 45° C., about35° C. to about 40° C., about 40° C. to about 50° C., about 40° C. toabout 45° C., or about 45° C. to about 50° C.).

In some embodiments, the biological sample can be incubated with apermeabilizing agent to facilitate permeabilization of the sample.Additional methods for sample permeabilization are described, forexample, in Jamur et al., Method Mol. Biol. 588:63-66, 2010, the entirecontents of which are incorporated herein by reference.

Lysis Reagents

In some embodiments, the biological sample can be permeabilized byadding one or more lysis reagents to the sample. Examples of suitablelysis agents include, but are not limited to, bioactive reagents such aslysis enzymes that are used for lysis of different cell types, e.g.,gram positive or negative bacteria, plants, yeast, mammalian, such aslysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase,and a variety of other commercially available lysis enzymes.

Other lysis agents can additionally or alternatively be added to thebiological sample to facilitate permeabilization. For example,surfactant-based lysis solutions can be used to lyse sample cells. Lysissolutions can include ionic surfactants such as, for example, sarcosyland sodium dodecyl sulfate (SDS). More generally, chemical lysis agentscan include, without limitation, organic solvents, chelating agents,detergents, surfactants, and chaotropic agents.

In some embodiments, the biological sample can be permeabilized bynon-chemical permeabilization methods. Non-chemical permeabilizationmethods are known in the art. For example, non-chemical permeabilizationmethods that can be used include, but are not limited to, physical lysistechniques such as electroporation, mechanical permeabilization methods(e.g., bead beating using a homogenizer and grinding balls tomechanically disrupt sample tissue structures), acousticpermeabilization (e.g., sonication), and thermal lysis techniques suchas heating to induce thermal permeabilization of the sample.

Proteases

In some embodiments, a medium, solution, or permeabilization solutionmay contain one or more proteases. In some embodiments, a biologicalsample treated with a protease capable of degrading histone proteins canresult in the generation of fragmented genomic DNA. The fragmentedgenomic DNA can be captured using the same capture domain (e.g., capturedomain having a poly(T) sequence) used to capture mRNA. In someembodiments, a biological sample is treated with a protease capable ofdegrading histone proteins and an RNA protectant prior to spatialprofiling in order to facilitate the capture of both genomic DNA andmRNA.

In some embodiments, a biological sample is permeabilized by exposingthe sample to a protease capable of degrading histone proteins. As usedherein, the term “histone protein” typically refers to a linker histoneprotein (e.g., H1) and/or a core histone protein (e.g., H2A, H2B, H3,and H4). In some embodiments, a protease degrades linker histoneproteins, core histone proteins, or linker histone proteins and corehistone proteins. Any suitable protease capable of degrading histoneproteins in a biological sample can be used. Non-limiting examples ofproteases capable of degrading histone proteins include proteasesinhibited by leupeptin and TLCK (Tosyl-L-lysyl-chloromethanehydrochloride), a protease encoded by the EUO gene from Chlamydiatrachomatis serovar A, granzyme A, a serine protease (e.g., trypsin ortrypsin-like protease, neutral serine protease, elastase, cathepsin G),an aspartyl protease (e.g., cathepsin D), a peptidase family C1 enzyme(e.g., cathepsin L), pepsin, proteinase K, a protease that is inhibitedby the diazomethane inhibitor Z-Phe-Phe-CHN(2) or the epoxide inhibitorE-64, a lysosomal protease, or an azurophilic enzyme (e.g., cathepsin G,elastase, proteinase 3, neutral serine protease). In some embodiments, aserine protease is a trypsin enzyme, trypsin-like enzyme or a functionalvariant or derivative thereof (e.g., P00761; C0HK48; Q8IYP2; Q8BW11;Q6IE06; P35035; P00760; P06871; Q90627; P16049; P07477; P00762; P35031;P19799; P35036; Q29463; P06872; Q90628; P07478; P07146; P00763; P35032;P70059; P29786; P35037; Q90629; P35030; P08426; P35033; P35038; P12788;P29787; P35039; P35040; Q8NHM4; P35041; P35043; P35044; P54624; P04814;P35045; P32821; P54625; P35004; P35046; P32822; P35047; C0HKA5; COHKA2;P54627; P35005; COHKA6; COHKA3; P52905; P83348; P00765; P35042; P81071;P35049; P51588; P35050; P35034; P35051; P24664; P35048; P00764; P00775;P54628; P42278; P54629; P42279; Q91041; P54630; P42280; C0HKA4) or acombination thereof. In some embodiments, a trypsin enzyme is P00761,P00760, Q29463, or a combination thereof. In some embodiments, aprotease capable of degrading one or more histone proteins comprises anamino acid sequence with at least 80% sequence identity to P00761,P00760, or Q29463. In some embodiments, a protease capable of degradingone or more histone proteins comprises an amino acid sequence with atleast 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identityto P00761, P00760, or Q29463. A protease may be considered a functionalvariant if it has at least 50% e.g., at least 60%, 70%, 80%, 90%, 95%,96%, 97%, 98%, 99%, or 100% of the activity relative to the activity ofthe protease in condition optimum for the enzyme. In some embodiments,the enzymatic treatment with pepsin enzyme, or pepsin like enzyme, caninclude: P03954/PEPA1_MACFU; P28712/PEPA1_RABIT; P27677/PEPA2_MACFU;P27821/PEPA2­_RABIT; P0DJD8/PEPA3_HUMAN; P27822/PEPA3_RABIT;P0DJD7/PEPA4_HUMAN; P27678/PEPA4_MACFU; P28713/PEPA4_RABIT;P0DJD9/PEPA5_HUMAN; Q9D106/PEPA5_MOUSE; P27823/PEPAF RABIT; P00792/PEPABOVIN; Q9N2D4/PEPA_CALJA; Q9GMY6/PEPA_CANLF; P00793/PEPA_CHICK;P11489/PEPA_MACMU; P00791/PEPA_PIG; Q9GMY7/PEPA_RHIFE;Q9GMY8/PEPA_SORUN; P81497/PEPA_SUNMU; P13636/PEPA_URSTH and functionalvariants and derivatives thereof, or a combination thereof. In someembodiments, the pepsin enzyme can include: P00791/PEPA_PIG;P00792/PEPA_BOVIN, functional variants, derivatives, or combinationsthereof.

Additionally, the protease may be contained in a reaction mixture(solution), which also includes other components (e.g., buffer, salt,chelator (e.g., EDTA), and/or detergent (e.g., SDS, N-Lauroylsarcosinesodium salt solution)). The reaction mixture may be buffered, having apH of about 6.5-8.5, e.g., about 7.0-8.0. Additionally, the reactionmixture may be used at any suitable temperature, such as about 10-50°C., e.g., about 10-44° C., 11-43° C., 12-42° C., 13-41° C., 14-40° C.,15-39° C., 16-38° C., 17-37° C., e.g., about 10° C., 12° C., 15° C., 18°C., 20° C., 22° C., 25° C., 28° C., 30° C., 33° C., 35° C. or 37° C.,preferably about 35-45° C., e.g., about 37° C.

Other Reagents

In some embodiments, a permeabilization solution can contain additionalreagents or a biological sample may be treated with additional reagentsin order to optimize biological sample permeabilization. In someembodiments, an additional reagent is an RNA protectant. As used herein,the term “RNA protectant” typically refers to a reagent that protectsRNA from RNA nucleases (e.g., RNases). Any appropriate RNA protectantthat protects RNA from degradation can be used. A non-limiting exampleof a RNA protectant includes organic solvents (e.g., at least 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95% v/v organic solvent), which include,without limitation, ethanol, methanol, propan-2-ol, acetone,trichloroacetic acid, propanol, polyethylene glycol, acetic acid, or acombination thereof. In some embodiments, a RNA protectant includesethanol, methanol and/or propan-2-ol, or a combination thereof. In someembodiments, a RNA protectant includes RNAlater ICE (ThermoFisherScientific). In some embodiments, the RNA protectant comprises at leastabout 60% ethanol. In some embodiments, the RNA protectant comprisesabout 60-95% ethanol, about 0-35% methanol and about 0-35% propan-2-ol,wherein the total amount of organic solvent in the medium is not morethan about 95%. In some embodiments, the RNA protectant comprises about60-95% ethanol, about 5-20% methanol and about 5-20% propan-2-ol,wherein the total amount of organic solvent in the medium is not morethan about 95%.

In some embodiments, the RNA protectant includes a salt. The salt mayinclude ammonium sulfate, ammonium bisulfate, ammonium chloride,ammonium acetate, cesium sulfate, cadmium sulfate, cesium iron (II)sulfate, chromium (III) sulfate, cobalt (II) sulfate, copper (II)sulfate, lithium chloride, lithium acetate, lithium sulfate, magnesiumsulfate, magnesium chloride, manganese sulfate, manganese chloride,potassium chloride, potassium sulfate, sodium chloride, sodium acetate,sodium sulfate, zinc chloride, zinc acetate and zinc sulfate. In someembodiments, the salt is a sulfate salt, for example, ammonium sulfate,ammonium bisulfate, cesium sulfate, cadmium sulfate, cesium iron (II)sulfate, chromium (III) sulfate, cobalt (II) sulfate, copper (II)sulfate, lithium sulfate, magnesium sulfate, manganese sulfate,potassium sulfate, sodium sulfate, or zinc sulfate. In some embodiments,the salt is ammonium sulfate. The salt may be present at a concentrationof about 20 g/100 ml of medium or less, such as about 15 g/100 ml, 10g/100 ml, 9 g/100 ml, 8 g/100 ml, 7 g/100 ml, 6 g/100 ml, 5 g/100 ml orless, e.g., about 4 g, 3 g, 2 g or 1 g/100 ml.

Additionally, the RNA protectant may be contained in a medium thatfurther includes a chelator (e.g., EDTA), a buffer (e.g., sodiumcitrate, sodium acetate, potassium citrate, or potassium acetate,preferably sodium acetate), and/or buffered to a pH between about 4-8(e.g., about 5).

In some embodiments, the biological sample is treated with one or moreRNA protectants before, contemporaneously with, or afterpermeabilization. For example, a biological sample is treated with oneor more RNA protectants prior to treatment with one or morepermeabilization reagents (e.g., one or more proteases). In anotherexample, a biological sample is treated with a solution including one ormore RNA protectants and one or more permeabilization reagents (e.g.,one or more proteases). In yet another example, a biological sample istreated with one or more RNA protectants after the biological sample hasbeen treated with one or more permeabilization reagents (e.g., one ormore proteases). In some embodiments, a biological sample is treatedwith one or more RNA protectants prior to fixation.

In some embodiments, identifying the location of the captured analyte inthe biological sample includes a nucleic acid extension reaction. Insome embodiments where a capture probe captures a fragmented genomic DNAmolecule, a nucleic acid extension reaction includes DNA polymerase. Forexample, a nucleic acid extension reaction includes using a DNApolymerase to extend the capture probe that is hybridized to thecaptured analyte (e.g., fragmented genomic DNA) using the capturedanalyte (e.g., fragmented genomic DNA) as a template. The product of theextension reaction includes a spatially-barcoded analyte (e.g.,spatially-barcoded fragmented genomic DNA). The spatially-barcodedanalyte (e.g., spatially-barcoded fragmented genomic DNA) can be used toidentify the spatial location of the analyte in the biological sample.Any DNA polymerase that is capable of extending the capture probe usingthe captured analyte as a template can be used for the methods describedherein. Non-limiting examples of DNA polymerases include T7 DNApolymerase; Bsu DNA polymerase; and E.coli DNA Polymerase pol I.

Diffusion-Resistant Media

In some embodiments, a diffusion-resistant medium, typically used tolimit diffusion of analytes, can include at least one permeabilizationreagent. For example, the diffusion-resistant medium (e.g., a hydrogel)can include wells (e.g., micro-, nano-, or picowells or pores)containing a permeabilization buffer or reagents. In some embodiments,the diffusion-resistant medium (e.g., a hydrogel) is soaked inpermeabilization buffer prior to contacting the hydrogel with a sample.In some embodiments, the hydrogel or other diffusion-resistant mediumcan contain dried reagents or monomers to deliver permeabilizationreagents when the diffusion-resistant medium is applied to a biologicalsample. In some embodiments, the diffusion-resistant medium, (e.g.,hydrogel) is covalently attached to a solid substrate (e.g., anacrylated glass slide).

In some embodiments, the hydrogel can be modified to both deliverpermeabilization reagents and contain capture. For example, a hydrogelfilm can be modified to include spatially-barcoded capture probes. Thespatially-barcoded hydrogel film is then soaked in permeabilizationbuffer before contacting the spatially-barcoded hydrogel film to thesample. In another example, a hydrogel can be modified to includespatially-barcoded capture probes and designed to serve as a porousmembrane (e.g., a permeable hydrogel) when exposed to permeabilizationbuffer or any other biological sample preparation reagent. Thepermeabilization reagent diffuses through the spatially-barcodedpermeable hydrogel and permeabilizes the biological sample on the otherside of the hydrogel. The analytes then diffuse into thespatially-barcoded hydrogel after exposure to permeabilization reagents.In such cases, the spatially-barcoded hydrogel (e.g., porous membrane)is facilitating the diffusion of the biological analytes in thebiological sample into the hydrogel. In some embodiments, biologicalanalytes diffuse into the hydrogel before exposure to permeabilizationreagents (e.g., when secreted analytes are present outside of thebiological sample or in instances where a biological sample is lysed orpermeabilized by other means prior to addition of permeabilizationreagents). In some embodiments, the permeabilization reagent is flowedover the hydrogel at a variable flow rate (e.g., any flow rate thatfacilitates diffusion of the permeabilization reagent across thespatially-barcoded hydrogel). In some embodiments, the permeabilizationreagents are flowed through a microfluidic chamber or channel over thespatially-barcoded hydrogel. In some embodiments, after using flow tointroduce permeabilization reagents to the biological sample, biologicalsample preparation reagents can be flowed over the hydrogel to furtherfacilitate diffusion of the biological analytes into thespatially-barcoded hydrogel. The spatially-barcoded hydrogel film thusdelivers permeabilization reagents to a sample surface in contact withthe spatially-barcoded hydrogel, enhancing analyte migration andcapture. In some embodiments, the spatially-barcoded hydrogel is appliedto a sample and placed in a permeabilization bulk solution. In someembodiments, the hydrogel film soaked in permeabilization reagents issandwiched between a sample and a spatially-barcoded array. In someembodiments, target analytes are able to diffuse through thepermeabilizing reagent soaked hydrogel and hybridize or bind the captureprobes on the other side of the hydrogel. In some embodiments, thethickness of the hydrogel is proportional to the resolution loss. Insome embodiments, wells (e.g., micro-, nano-, or picowells) can containspatially-barcoded capture probes and permeabilization reagents and/orbuffer. In some embodiments, spatially-barcoded capture probes andpermeabilization reagents are held between spacers. In some embodiments,the sample is punch, cut, or transferred into the well, wherein a targetanalyte diffuses through the permeabilization reagent/buffer and to thespatially-barcoded capture probes. In some embodiments, resolution lossmay be proportional to gap thickness (e.g., the amount ofpermeabilization buffer between the sample and the capture probes). Insome embodiments, the diffusion-resistant medium (e.g., hydrogel) isbetween approximately 50-500 micrometers thick including 500, 450, 400,350, 300, 250, 200, 150, 100, or 50 micrometers thick, or any thicknesswithin 50 and 500 micrometers.

In some embodiments, a biological sample is exposed to a porous membrane(e.g., a permeable hydrogel) to aid in permeabilization and limitdiffusive analyte losses, while allowing permeabilization reagents toreach a sample. Membrane chemistry and pore volume can be manipulated tominimize analyte loss. In some embodiments, the porous membrane may bemade of glass, silicon, paper, hydrogel, polymer monoliths, or othermaterial. In some embodiments, the material may be naturally porous. Insome embodiments, the material may have pores or wells etched into solidmaterial. In some embodiments, the permeabilization reagents are flowedthrough a microfluidic chamber or channel over the porous membrane. Insome embodiments, the flow controls the sample’s access to thepermeabilization reagents. In some embodiments, the porous membrane is apermeable hydrogel. For example, a hydrogel is permeable whenpermeabilization reagents and/or biological sample preparation reagentscan pass through the hydrogel using diffusion. Any suitablepermeabilization reagents and/or biological sample preparation reagentsdescribed herein can be used under conditions sufficient to releaseanalytes (e.g., nucleic acid, protein, metabolites, lipids, etc.) fromthe biological sample. In some embodiments, a hydrogel is exposed to thebiological sample on one side and permeabilization reagent on the otherside. The permeabilization reagent diffuses through the permeablehydrogel and permeabilizes the biological sample on the other side ofthe hydrogel. In some embodiments, permeabilization reagents are flowedover the hydrogel at a variable flow rate (e.g., any flow rate thatfacilitates diffusion of the permeabilization reagent across thehydrogel). In some embodiments, the permeabilization reagents are flowedthrough a microfluidic chamber or channel over the hydrogel. Flowingpermeabilization reagents across the hydrogel enables control of theconcentration of reagents. In some embodiments, hydrogel chemistry andpore volume can be tuned to enhance permeabilization and limit diffusiveanalyte losses.

In some embodiments, a porous membrane is sandwiched between aspatially-barcoded array and the sample, wherein permeabilizationsolution is applied over the porous membrane. The permeabilizationreagents diffuse through the pores of the membrane and into thebiological sample. In some embodiments, the biological sample can beplaced on a substrate (e.g., a glass slide). Biological analytes thendiffuse through the porous membrane and into to the space containing thecapture probes. In some embodiments, the porous membrane is modified toinclude capture probes. For example, the capture probes can be attachedto a surface of the porous membrane using any of the methods describedherein. In another example, the capture probes can be embedded in theporous membrane at any depth that allows interaction with a biologicalanalyte. In some embodiments, the porous membrane is placed onto abiological sample in a configuration that allows interaction between thecapture probes on the porous membrane and the biological analytes fromthe biological sample. For example, the capture probes are located onthe side of the porous membrane that is proximal to the biologicalsample. In such cases, permeabilization reagents on the other side ofthe porous membrane diffuse through the porous membrane into thelocation containing the biological sample and the capture probes inorder to facilitate permeabilization of the biological sample (e.g.,also facilitating capture of the biological analytes by the captureprobes). In some embodiments, the porous membrane is located between thesample and the capture probes. In some embodiments, the permeabilizationreagents are flowed through a microfluidic chamber or channel over theporous membrane.

Selective Permeabilization/Selective Lysis

In some embodiments, biological samples can be processed to selectivelyrelease an analyte from a subcellular region of a cell according toestablished methods. In some embodiments, a method provided herein caninclude detecting at least one biological analyte present in asubcellular region of a cell in a biological sample. As used herein, a“subcellular region” can refer to any subcellular region. For example, asubcellular region can refer to cytosol, a mitochondria, a nucleus, anucleolus, an endoplasmic reticulum, a lysosome, a vesicle, a Golgiapparatus, a plastid, a vacuole, a ribosome, cytoskeleton, orcombinations thereof. In some embodiments, the subcellular regioncomprises at least one of cytosol, a nucleus, a mitochondria, and amicrosome. In some embodiments, the subcellular region is cytosol. Insome embodiments, the subcellular region is a nucleus. In someembodiments, the subcellular region is a mitochondria. In someembodiments, the subcellular region is a microsome.

For example, a biological analyte can be selectively released from asubcellular region of a cell by selective permeabilization or selectivelysing. In some embodiments, “selective permeabilization” can refer to apermeabilization method that can permeabilize a membrane of asubcellular region while leaving a different subcellular regionsubstantially intact (e.g., biological analytes are not released fromsubcellular region due to the applied permeabilization method).Non-limiting examples of selective permeabilization methods includeusing electrophoresis and/or applying a permeabilization reagent. Insome embodiments, “selective lysing” can refer to a lysis method thatcan lyse a membrane of a subcellular region while leaving a differentsubcellular region substantially intact (e.g., biological analytes arenot released from subcellular region due to the applied lysis method).Several methods for selective permeabilization or lysis are known to oneof skill in the art including the methods described in Lu et al. LabChip. 2005 Jan;5(1):23-9; Niklas et al. Anal Biochem. 2011 Sep15;416(2):218-27; Cox and Emili. Nat Protoc. 2006;1(4):1872-8; Chiang etal. J Biochem. Biophys. Methods. 2000 Nov 20;46(1-2):53-68; and Yamauchiand Herr et al. Microsyst. Nanoeng. 2017;3. pii: 16079; each of which isincorporated herein by reference in its entirety.

In some embodiments, “selective permeabilization” or “selective lysis”refer to the selective permeabilization or selective lysis of a specificcell type. For example, “selective permeabilization” or “selectivelysis” can refer to lysing one cell type while leaving a different celltype substantially intact (e.g., biological analytes are not releasedfrom the cell due to the applied permeabilization or lysis method). Acell that is a “different cell type” than another cell can refer to acell from a different taxonomic kingdom, a prokaryotic cell versus aeukaryotic cell, a cell from a different tissue type, etc. Many methodsare known to one of skill in the art for selectively permeabilizing orlysing different cell types. Non-limiting examples include applying apermeabilization reagent, electroporation, and/or sonication. See, e.g.,International Application No. WO 2012/168003; Han et al. MicrosystNanoeng. 2019 Jun 17;5:30; Gould et al. Oncotarget. 2018 Mar 20; 9(21):15606-15615; Oren and Shai. Biochemistry. 1997 Feb 18;36(7):1826-35;Algayer et al. Molecules. 2019 May 31;24(11). pii: E2079; Hipp et al.Leukemia. 2017 Oct;31(10):2278; International Application No. WO2012/168003; and U.S. Pat. No. 7,785,869; all of which are incorporatedby reference herein in their entireties.

In some embodiments, applying a selective permeabilization or lysisreagent comprises contacting the biological sample with a hydrogelcomprising the permeabilization or lysis reagent.

In some embodiments, the biological sample is contacted with two or morearrays (e.g., flexible arrays, as described herein). For example, aftera subcellular region is permeabilized and a biological analyte from thesubcellular region is captured on a first array, the first array can beremoved, and a biological analyte from a different subcellular regioncan be captured on a second array.

Selective Enrichment of RNA Species

In some embodiments, where RNA is the analyte, one or more RNA analytespecies of interest can be selectively enriched (e.g., Adiconis, et.al., Comparative analysis of RNA sequencing methods for degraded andlow-input samples, Nature, vol. 10, July 2013, 623-632, hereinincorporated by reference in its entirety). For example, one or morespecies of RNA can be selected by addition of one or moreoligonucleotides to the sample. In some embodiments, the additionaloligonucleotide is a sequence used for priming a reaction by apolymerase. For example, one or more primer sequences with sequencecomplementarity to one or more RNAs of interest can be used to amplifythe one or more RNAs of interest, thereby selectively enriching theseRNAs. In some embodiments, an oligonucleotide with sequencecomplementarity to the complementary strand of captured RNA (e.g., cDNA)can bind to the cDNA. For example, biotinylated oligonucleotides withsequence complementary to one or more cDNAs of interest binds to thecDNA and can be selected using biotinylation-streptavidin affinity usingany of a variety of methods known to the field (e.g., streptavidinbeads).

Alternatively, one or more species of RNA (e.g., ribosomal and/ormitochondrial RNA) can be down-selected (e.g., removed, depleted) usingany of a variety of methods. Non-limiting examples of a hybridizationand capture method of ribosomal RNA depletion include RiboMinus™,RiboCop™, and Ribo-Zero™. Another non-limiting RNA depletion methodinvolves hybridization of complementary DNA oligonucleotides to unwantedRNA followed by degradation of the RNA/DNA hybrids using RNase H.Non-limiting examples of a hybridization and degradation method includeNEBNext® rRNA depletion, NuGEN AnyDeplete, TruSeq™. Another non-limitingribosomal RNA depletion method includes ZapR™ digestion, for exampleSMARTer. In the SMARTer method, random nucleic acid adapters arehybridized to RNA for first-strand synthesis and tailing by reversetranscriptase, followed by template switching and extension by reversetranscriptase. Additionally, first round PCR amplification addsfull-length Illumina sequencing adapters (e.g., Illumina indexes).Ribosomal RNA is cleaved by ZapR v2 and R probes v2. A second round ofPCR is performed, amplifying non-rRNA molecules (e.g., cDNA). Parts orsteps of these ribosomal depletion protocols/kits can be furthercombined with the methods described herein to optimize protocols for aspecific biological sample.

In depletion protocols, probes can be administered to a sample thatselectively hybridize to ribosomal RNA (rRNA), thereby reducing the pooland concentration of rRNA in the sample. Probes can be administered to abiological sample that selectively hybridize to mitochondria RNA(mtRNA), thereby reducing the pool and concentration of mtRNA in thesample. In some embodiments, probes complementary to mitochondrial RNAcan be added during cDNA synthesis, or probes complementary to bothribosomal and mitochondrial RNA can be added during cDNA synthesis.Subsequent application of capture probes to the sample can result inimproved capture of other types of RNA due to a reduction innon-specific RNA (e.g., down-selected RNA) present in the sample.Additionally and alternatively, duplex-specific nuclease (DSN) treatmentcan remove rRNA (see, e.g., Archer, et al, Selective and flexibledepletion of problematic sequences from RNA-seq libraries at the cDNAstage, BMC Genomics, 15 401, (2014), the entire contents of which areincorporated herein by reference). Furthermore, hydroxyapatitechromatography can remove abundant species (e.g., rRNA) (see, e.g.,Vandernoot, V.A., cDNA normalization by hydroxyapatite chromatography toenrich transcriptome diversity in RNA-seq applications, Biotechniques,53(6) 373-80, (2012), the entire contents of which are incorporatedherein by reference).

Other Reagents

Additional reagents can be added to a biological sample to performvarious functions prior to analysis of the biological sample. In someembodiments, nuclease inhibitors such as DNase and RNase inactivatingagents or protease inhibitors, and/or chelating agents such as EDTA, canbe added to the biological sample. In other embodiments nucleases, suchas DNase or RNAse, or proteases, such as pepsin or proteinase K, areadded to the sample. In some embodiments, additional reagents may bedissolved in a solution or applied as a medium to the sample. In someembodiments, additional reagents (e.g., pepsin) may be dissolved in HClprior to applying to the sample.

In some embodiments, the biological sample can be treated with one ormore enzymes. For example, one or more endonucleases to fragment DNA,DNA polymerase enzymes, and dNTPs used to amplify nucleic acids can beadded. Other enzymes that can also be added to the biological sampleinclude, but are not limited to, polymerase, transposase, ligase, andDNAse, and RNAse.

In some embodiments, reverse transcriptase enzymes can be added to thesample, including enzymes with terminal transferase activity, primers,and template switch oligonucleotides (TSOs). Template switching can beused to increase the length of a cDNA, e.g., by appending a predefinednucleic acid sequence to the cDNA. In some embodiments, the appendednucleic acid sequence comprises one or more ribonucleotides.

In some embodiments, additional reagents can be added to improve therecovery of one or more target molecules (e.g., cDNA molecules, mRNAtranscripts). For example, addition of carrier RNA to a RNA sampleworkflow process can increase the yield of extracted RNA/DNA hybridsfrom the biological sample. In some embodiments, carrier molecules areuseful when the concentration of input or target molecules is low ascompared to remaining molecules. Generally, single target moleculescannot form a precipitate, and addition of the carrier molecules canhelp in forming a precipitate. Some target molecule recovery protocolsuse carrier RNA to prevent small amounts of target nucleic acids presentin the sample from being irretrievably bound. In some embodiments,carrier RNA can be added immediately prior to a second strand synthesisstep. In some embodiments, carrier RNA can be added immediately prior toa second strand cDNA synthesis on oligonucleotides released from anarray. In some embodiments, carrier RNA can be added immediately priorto a post in vitro transcription clean-up step. In some embodiments,carrier RNA can be added prior to amplified RNA purification andquantification. In some embodiments, carrier RNA can be added before RNAquantification. In some embodiments, carrier RNA can be addedimmediately prior to both a second strand cDNA synthesis and a post invitro transcription clean-up step.

Pre-processing for Capture Probe Interaction

In some embodiments, analytes in a biological sample can bepre-processed prior to interaction with a capture probe. For example,prior to interaction with capture probes, polymerization reactionscatalyzed by a polymerase (e.g., DNA polymerase or reversetranscriptase) are performed in the biological sample. In someembodiments, a primer for the polymerization reaction includes afunctional group that enhances hybridization with the capture probe. Thecapture probes can include appropriate capture domains to capturebiological analytes of interest (e.g., poly(dT) sequence to capturepoly(A) mRNA).

In some embodiments, biological analytes are pre-processed for librarygeneration via next generation sequencing. For example, analytes can bepre-processed by addition of a modification (e.g., ligation of sequencesthat allow interaction with capture probes). In some embodiments,analytes (e.g., DNA or RNA) are fragmented using fragmentationtechniques (e.g., using transposases and/or fragmentation buffers).

Fragmentation can be followed by a modification of the analyte. Forexample, a modification can be the addition through ligation of anadapter sequence that allows hybridization with the capture probe. Insome embodiments, where the analyte of interest is RNA, poly(A) tailingis performed. Addition of a poly(A) tail to RNA that does not contain apoly(A) tail can facilitate hybridization with a capture probe thatincludes a capture domain with a functional amount of poly(dT) sequence.

In some embodiments, prior to interaction with capture probes, ligationreactions catalyzed by a ligase are performed in the biological sample.In some embodiments, ligation can be performed by chemical ligation. Insome embodiments, the ligation can be performed using click chemistry asfurther described below. In some embodiments, the capture domainincludes a DNA sequence that has complementarity to a RNA molecule,where the RNA molecule has complementarity to a second DNA sequence, andwhere the RNA-DNA sequence complementarity is used to ligate the secondDNA sequence to the DNA sequence in the capture domain. In theseembodiments, direct detection of RNA molecules is possible.

In some embodiments, prior to interaction with capture probes,target-specific reactions are performed in the biological sample.Examples of target specific reactions include, but are not limited to,ligation of target specific adaptors, probes and/or otheroligonucleotides, target specific amplification using primers specificto one or more analytes, and target-specific detection using in situhybridization, DNA microscopy, and/or antibody detection. In someembodiments, a capture probe includes capture domains targeted totarget-specific products (e.g., amplification or ligation).

II. General Spatial Array-Based Analytical Methodology

Provided herein are methods, apparatus, systems, and compositions forspatial array-based analysis of biological samples.

(A) Spatial Analysis Methods

Array-based spatial analysis methods involve the transfer of one or moreanalytes from a biological sample to an array of features on asubstrate, where each feature is associated with a unique spatiallocation on the array. Subsequent analysis of the transferred analytesincludes determining the identity of the analytes and the spatiallocation of each analyte within the biological sample. The spatiallocation of each analyte within the biological sample is determinedbased on the feature to which each analyte is bound on the array, andthe feature’s relative spatial location within the array.

There are at least two general methods to associate a spatial barcodewith one or more neighboring cells, such that the spatial barcodeidentifies the one or more cells, and/or contents of the one or morecells, as associated with a particular spatial location. One generalmethod is to promote analytes out of a cell and towards thespatially-barcoded array. FIG. 1 depicts an exemplary embodiment of thisgeneral method. In FIG. 1 , the spatially-barcoded array populated withcapture probes (as described further herein) is contacted with abiological sample 101, and biological sample is permeabilized, allowingthe analyte to migrate away from the sample and toward the array. Theanalyte interacts with a capture probe on the spatially-barcoded array102. Once the analyte hybridizes/is bound to the capture probe, thesample is optionally removed from the array and the capture probes areanalyzed in order to obtain spatially-resolved analyte information 103.

Another general method is to cleave the spatially-barcoded captureprobes from an array, and promote the spatially-barcoded capture probestowards and/or into or onto the biological sample. FIG. 2 depicts anexemplary embodiment of this general method, the spatially-barcodedarray populated with capture probes (as described further herein) can becontacted with a sample 201. The spatially-barcoded capture probes arecleaved and then interact with cells within the provided biologicalsample 202. The interaction can be a covalent or non-covalentcell-surface interaction. The interaction can be an intracellularinteraction facilitated by a delivery system or a cell penetrationpeptide. Once the spatially-barcoded capture probe is associated with aparticular cell, the sample can be optionally removed for analysis. Thesample can be optionally dissociated before analysis. Once the taggedcell is associated with the spatially-barcoded capture probe, thecapture probes can be analyzed to obtain spatially-resolved informationabout the tagged cell 203.

FIG. 3 shows an exemplary workflow that includes preparing a biologicalsample on a spatially-barcoded array 301. Sample preparation may includeplacing the sample on a slide, fixing the sample, and/or staining thebiological sample for imaging. The stained sample can be then imaged onthe array 302 using both brightfield (to image the sample hematoxylinand eosin stain) and fluorescence (to image features) modalities.Optionally, the sample can be destained prior to permeabilization. Insome embodiments, analytes are then released from the sample and captureprobes forming the spatially-barcoded array hybridize or bind thereleased analytes 303. The sample is then removed from the array 304 andthe capture probes cleaved from the array 305. The biological sample andarray are then optionally imaged a second time in both modalities 305Bwhile the analytes are reverse transcribed into cDNA, and an ampliconlibrary is prepared 306 and sequenced 307. The two sets of images arethen spatially-overlaid in order to correlate spatially-identifiedbiological sample information 308. When the sample and array are notimaged a second time, 305B, a spot coordinate file is supplied instead.The spot coordinate file replaces the second imaging step 305B. Further,amplicon library preparation 306 can be performed with a unique PCRadapter and sequenced 307.

FIG. 4 shows another exemplary workflow that utilizes aspatially-barcoded array on a substrate, where spatially-barcodedcapture probes are clustered at areas called features. Thespatially-barcoded capture probes can include a cleavage domain, one ormore functional domains, a spatial barcode, a unique molecularidentifier, and a capture domain. The spatially-barcoded capture probescan also include a 5′ end modification for reversible attachment to thesubstrate. The spatially-barcoded array is contacted with a biologicalsample 401, and the sample is permeabilized through application ofpermeabilization reagents 402. Permeabilization reagents may beadministered by placing the array/sample assembly within a bulksolution. Alternatively, permeabilization reagents may be administeredto the sample via a diffusion-resistant medium and/or a physical barriersuch as a lid, wherein the sample is sandwiched between thediffusion-resistant medium and/or barrier and the array-containingsubstrate. The analytes are migrated toward the spatially-barcodedcapture array using any number of techniques disclosed herein. Forexample, analyte migration can occur using a diffusion-resistant mediumlid and passive migration. As another example, analyte migration can beactive migration, using an electrophoretic transfer system, for example.Once the analytes are in close proximity to the spatially-barcodedcapture probes, the capture probes can hybridize or otherwise bind atarget analyte 403. The biological sample can be optionally removed fromthe array 404.

The capture probes can be optionally cleaved from the array 405, and thecaptured analytes can be spatially-barcoded by performing a reversetranscriptase first strand cDNA reaction. A first strand cDNA reactioncan be optionally performed using template switching oligonucleotides.For example, a template switching oligonucleotide can hybridize to apoly(C) tail added to a 3′end of the cDNA by a reverse transcriptaseenzyme in a template independent manner. The original mRNA template andtemplate switching oligonucleotide can then be denatured from the cDNAand the spatially-barcoded capture probe can then hybridize with thecDNA and a complement of the cDNA can be generated. The first strandcDNA can then be purified and collected for downstream amplificationsteps. The first strand cDNA can be amplified using PCR 406, where theforward and reverse primers flank the spatial barcode and analyteregions of interest, generating a library associated with a particularspatial barcode. In some embodiments, the cDNA comprises a sequencing bysynthesis (SBS) primer sequence. The library amplicons are sequenced andanalyzed to decode spatial information 407.

Using the methods, compositions, systems, kits, and devices describedherein, RNA transcripts present in biological samples (e.g., tissuesamples) can be used for spatial transcriptome analysis. In particular,in some cases, the barcoded oligonucleotides may be configured to prime,replicate, and consequently yield barcoded extension products from anRNA template, or derivatives thereof. For example, in some cases, thebarcoded oligonucleotides may include mRNA specific priming sequences,e.g., poly-T primer segments that allow priming and replication of mRNAin a reverse transcription reaction or other targeted priming sequences.Alternatively or additionally, random RNA priming may be carried outusing random N-mer primer segments of the barcoded oligonucleotides.Reverse transcriptases (RTs) can use an RNA template and a primercomplementary to the 3′ end of the RNA template to direct the synthesisof the first strand complementary DNA (cDNA). Many RTs can be used inthis reverse transcription reactions, including, for example, avianmyeloblastosis virus (AMV) reverse transcriptase, moloney murineleukemia virus (M-MuLV or MMLV), and other variants thereof. Somerecombinant M-MuLV reverse transcriptase, such as, for example,PROTOSCRIPT® II reverse transcriptase, can have reduced RNase H activityand increased thermostability when compared to its wild typecounterpart, and provide higher specificity, higher yield of cDNA andmore full-length cDNA products with up to 12 kilobase (kb) in length. Insome embodiments, the reverse transcriptase enzyme is a mutant reversetranscriptase enzyme such as, but not limited to, mutant MMLV reversetranscriptase. In another embodiment, the reverse transcriptase is amutant MMLV reverse transcriptase such as, but not limited to, one ormore variants described in US Pat. Publication No. 20180312822, which isincorporated herein by reference in its entirety.

FIG. 5 depicts an exemplary workflow where the biological sample isremoved from the spatially-barcoded array and the spatially-barcodedcapture probes are removed from the array for barcoded analyteamplification and library preparation. Another embodiment includesperforming first strand synthesis using template switchingoligonucleotides on the spatially-barcoded array without cleaving thecapture probes. In this embodiment, sample preparation 501 andpermeabilization 502 are performed as described elsewhere herein. Oncethe capture probes capture the analyte(s), first strand cDNA created bytemplate switching and reverse transcriptase 503 is then denatured andthe second strand is then extended 504. The second strand cDNA is thendenatured from the first strand cDNA, neutralized, and transferred to atube 505. cDNA quantification and amplification can be performed usingstandard techniques discussed herein. The cDNA can then be subjected tolibrary preparation 506 and indexing 507, including fragmentation,end-repair, and a-tailing, and indexing PCR steps.

In a non-limiting example of the workflows described above, a biologicalsample (e.g., tissue section), can be fixed with methanol, stained withhematoxylin and eosin, and imaged. Optionally, the sample can bedestained prior to permeabilization. The images can be used to mapspatial gene expression patterns back to the biological sample. Apermeabilization enzyme can be used to permeabilize the biologicalsample directly on the slide. Analytes (e.g., polyadenylated mRNA)released from the overlying cells of the biological sample can becaptured by capture probes within a capture area on a substrate. Reversetranscription (RT) reagents can be added to permeabilized biologicalsamples. Incubation with the RT reagents can produce spatially-barcodedfull-length cDNA from the captured analytes (e.g., polyadenylated mRNA).Second strand reagents (e.g., second strand primers, enzymes) can beadded to the biological sample on the slide to initiate second strandsynthesis. The resulting cDNA can be denatured from the capture probetemplate and transferred (e.g., to a clean tube) for amplification,and/or library construction. The spatially-barcoded, full-length cDNAcan be amplified via PCR prior to library construction. The cDNA canthen be enzymatically fragmented and size-selected in order to optimizethe cDNA amplicon size. P5, P7, i7, and i5 can be used as sampleindexes, and TruSeq Read 2 can be added via End Repair, A-tailing,Adaptor Ligation, and PCR. The cDNA fragments can then be sequencedusing paired-end sequencing using TruSeq Read 1 and TruSeq Read 2 assequencing primer sites.

In some embodiments, performing correlative analysis of data produced bythis workflow, and other workflows described herein, can yield over 95%correlation of genes expressed across two capture areas (e.g., 95% orgreater, 96% or greater, 97% or greater, 98% or greater, or 99% orgreater). When performing the described workflows using single cell RNAsequencing of nuclei, in some embodiments, correlative analysis of thedata can yield over 90% (e.g., over 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99%) correlation of genes expressed across two captureareas.

In some embodiments, after cDNA is generated (e.g., by reversetranscription) the cDNA can be amplified directly on the substratesurface. Generating multiple copies of the cDNA (e.g., cDNA synthesizedfrom captured analytes) via amplification directly on the substratesurface can improve final sequencing library complexity. Thus, in someembodiments, cDNA can be amplified directly on the substrate surface byisothermal nucleic acid amplification. In some embodiments, isothermalnucleic acid amplification can amplify RNA or DNA

In some embodiments, isothermal amplification can be faster than astandard PCR reaction. In some embodiments, isothermal amplification canbe linear amplification (e.g., asymmetrical with a single primer), orexponential amplification (e.g., with two primers). In some embodiments,isothermal nucleic acid amplification can be performed by atemplate-switching oligonucleotide primer. In some embodiments, thetemplate switching oligonucleotide adds a common sequence onto the 5′end of the RNA being reverse transcribed. For example, after a captureprobe interacts with an analyte (e.g., mRNA) and reverse transcriptionis performed such that additional nucleotides are added to the end ofthe cDNA creating a 3′ overhang as described herein. In someembodiments, a template switching oligonucleotide hybridizes tountemplated poly(C) nucleotides added by a reverse transcriptase tocontinue replication to the 5′ end of the template switchingoligonucleotide, thereby generating full-length cDNA ready for furtheramplification. In some embodiments, the template switchingoligonucleotide adds a common 5′ sequence to full-length cDNA that isused for cDNA amplification (e.g., a reverse complement of the templateswitching oligonucleotide).

In some embodiments, once a full-length cDNA molecule is generated, thetemplate switching oligonucleotide can serve as a primer in a cDNAamplification reaction (e.g., with a DNA polymerase). In someembodiments, double stranded cDNA (e.g., first strand cDNA and secondstrand reverse complement cDNA) can be amplified via isothermalamplification with either a helicase or recombinase, followed by astrand displacing DNA polymerase. The strand displacing DNA polymerasecan generate a displaced second strand resulting in an amplifiedproduct.

In any of isothermal amplification methods described herein, barcodeexchange (e.g., spatial barcode) can occur after the first amplificationcycle where there are unused capture probes on the substrate surface. Insome embodiments, the free 3′OH end of the unused capture probes can beblocked by any suitable 3′OH blocking method. In some embodiments, the3′OH can be blocked by hairpin ligation.

Isothermal nucleic acid amplification can be used in addition to, or asan alternative to standard PCR reactions (e.g., a PCR reaction thatrequires heating to about 95° C. to denature double stranded DNA).Isothermal nucleic acid amplification generally does not require the useof a thermocycler, however in some embodiments, isothermal amplificationcan be performed in a thermocycler. In some embodiments, isothermalamplification can be performed from about 35° C. to about 75° C. In someembodiments, isothermal amplification can be performed from about 40°C., about 45° C., about 50° C., about 55° C., about 60° C., about 65°C., or about 70° C. or anywhere in between depending on the polymeraseand auxiliary enzymes used.

Isothermal nucleic acid amplification techniques are known in the art,and can be used alone or in combination with any of the spatial methodsdescribed herein. For example, non-limiting examples of suitableisothermal nucleic acid amplification techniques include transcriptionmediated amplification, nucleic acid sequence-based amplification,signal mediated amplification of RNA technology, strand displacementamplification, rolling circle amplification, loop-mediated isothermalamplification of DNA (LAMP), isothermal multiple displacementamplification, recombinase polymerase amplification, helicase-dependentamplification, single primer isothermal amplification, and circularhelicase-dependent amplification (See, e.g., Gill and Ghaemi, Nucleicacid isothermal amplification technologies: a review, Nucleosides,Nucleotides, & Nucleic Acids, 27(3), 224-43, doi:10.1080/15257770701845204 (2008), which is incorporated herein byreference in its entirety).

In some embodiments, the isothermal nucleic acid amplification ishelicase-dependent nucleic acid amplification. Helicase-dependentisothermal nucleic acid amplification is described in Vincent, et. al.,Helicase-dependent isothermal DNA amplification, EMBO Rep., 795-800(2004) and U.S. Pat. No. 7,282,328, which are both incorporated hereinby reference in their entireties. Further, helicase-dependent nucleicacid amplification on a substrate (e.g., on-chip) is described inAndresen, et. al., Helicase-dependent amplification: use in OnChipamplification and potential for point-of-care diagnostics, Expert RevMol Diagn., 9, 645-650, doi: 10.1586/erm.09.46 (2009), which isincorporated herein by reference in its entirety. In some embodiments,the isothermal nucleic acid amplification is recombinase polymerasenucleic acid amplification. Recombinase polymerase nucleic acidamplification is described in Piepenburg, et al., DNA Detection UsingRecombinant Proteins, PLoS Biol., 4, 7 e204 (2006) and Li, et. al.,Review: a comprehensive summary of a decade development of therecombinase polymerase amplification, Analyst, 144, 31-67, doi:10.1039/C8AN01621F (2019), both of which are incorporated herein byreference in their entireties.

Generally, isothermal amplification techniques use standard PCR reagents(e.g., buffer, dNTPs etc.) known in the art. Some isothermalamplification techniques can require additional reagents. For example,helicase dependent nucleic acid amplification uses a single-strandbinding protein and an accessory protein. In another example,recombinase polymerase nucleic acid amplification uses recombinase(e.g., T4 UvsX), recombinase loading factor (e.g., TF UvsY),single-strand binding protein (e.g., T4 gp32), crowding agent (e.g.,PEG-35K), and ATP.

After isothermal nucleic acid amplification of the full-length cDNAdescribed by any of the methods herein, the isothermally amplified cDNAs(e.g., single-stranded or double-stranded) can be recovered from thesubstrate, and optionally followed by amplification with typical cDNAPCR in microcentrifuge tubes. The sample can then be used with any ofthe spatial methods described herein.

(I) Immunohistochemistry and Immunofluorescence

In some embodiments, immunofluorescence or immunohistochemistryprotocols (direct and indirect staining techniques) can be performed asa part of, or in addition to, the exemplary spatial workflows presentedherein. For example, tissue sections can be fixed according to methodsdescribed herein. The biological sample can be transferred to an array(e.g., capture probe array), wherein analytes (e.g., proteins) areprobed using immunofluorescence protocols. For example, the sample canbe rehydrated, blocked, and permeabilized (3XSSC, 2% BSA, 0.1% Triton X,1 U/µl RNAse inhibitor for 10 min at 4° C.) before being stained withfluorescent primary antibodies (1:100 in 3XSSC, 2% BSA, 0.1% Triton X, 1U/µl RNAse inhibitor for 30 min at 4° C.). The biological sample can bewashed, coverslipped (in glycerol + 1 U/µl RNAse inhibitor), imaged(e.g., using a confocal microscope or other apparatus capable offluorescent detection), washed, and processed according to analytecapture or spatial workflows described herein.

As used herein, an “antigen retrieval buffer” can improve antibodycapture in IF/IHC protocols. An exemplary protocol for antigen retrievalcan be preheating the antigen retrieval buffer (e.g., to 95° C.),immersing the biological sample in the heated antigen retrieval bufferfor a predetermined time, and then removing the biological sample fromthe antigen retrieval buffer and washing the biological sample.

In some embodiments, optimizing permeabilization can be useful foridentifying intracellular analytes. Permeabilization optimization caninclude selection of permeabilization agents, concentration ofpermeabilization agents, and permeabilization duration. Tissuepermeabilization is discussed elsewhere herein.

In some embodiments, blocking an array and/or a biological sample inpreparation of labeling the biological sample decreases unspecificbinding of the antibodies to the array and/or biological sample(decreases background). Some embodiments provide for blockingbuffers/blocking solutions that can be applied before and/or duringapplication of the label, wherein the blocking buffer can include ablocking agent, and optionally a surfactant and/or a salt solution. Insome embodiments, a blocking agent can be bovine serum albumin (BSA),serum, gelatin (e.g., fish gelatin), milk (e.g., non-fat dry milk),casein, polyethylene glycol (PEG), polyvinyl alcohol (PVA), orpolyvinylpyrrolidone (PVP), biotin blocking reagent, a peroxidaseblocking reagent, levamisole, Carnoy’s solution, glycine, lysine, sodiumborohydride, pontamine sky blue, Sudan Black, trypan blue, FITC blockingagent, and/or acetic acid. The blocking buffer/blocking solution can beapplied to the array and/or biological sample prior to and/or duringlabeling (e.g., application of fluorophore-conjugated antibodies) to thebiological sample.

In some embodiments, additional steps or optimizations can be includedin performing IF/IHC protocols in conjunction with spatial arrays.Additional steps or optimizations can be included in performingspatially-tagged analyte capture agent workflows discussed herein.

In some embodiments, provided herein are methods for spatially detectingan analyte (e.g., detecting the location of an analyte, e.g., abiological analyte) from a biological sample (e.g., an analyte presentin a biological sample, such as a tissue section) that include: (a)providing a biological sample on a substrate; (b) staining thebiological sample on the substrate, imaging the stained biologicalsample, and selecting the biological sample or subsection of thebiological sample (e.g., region of interest) to subject to analysis; (c)providing an array comprising one or more pluralities of capture probeson a substrate; (d) contacting the biological sample with the array,thereby allowing a capture probe of the one or more pluralities ofcapture probes to capture the analyte of interest; and (e) analyzing thecaptured analyte, thereby spatially detecting the analyte of interest.Any variety of staining and imaging techniques as described herein orknown in the art can be used in accordance with methods describedherein. In some embodiments, the staining includes optical labels asdescribed herein, including, but not limited to, fluorescent,radioactive, chemiluminescent, calorimetric, or colorimetric detectablelabels. In some embodiments, the staining includes a fluorescentantibody directed to a target analyte (e.g., cell surface orintracellular proteins) in the biological sample. In some embodiments,the staining includes an immunohistochemistry stain directed to a targetanalyte (e.g., cell surface or intracellular proteins) in the biologicalsample. In some embodiments, the staining includes a chemical stain suchas hematoxylin and eosin (H&E) or periodic acid-schiff (PAS). In someembodiments, significant time (e.g., days, months, or years) can elapsebetween staining and/or imaging the biological sample and performinganalysis. In some embodiments, reagents for performing analysis areadded to the biological sample before, contemporaneously with, or afterthe array is contacted to the biological sample. In some embodiments,step (d) includes placing the array onto the biological sample. In someembodiments, the array is a flexible array where the plurality ofspatially-barcoded features (e.g., a substrate with capture probes, abead with capture probes) are attached to a flexible substrate. In someembodiments, measures are taken to slow down a reaction (e.g., coolingthe temperature of the biological sample or using enzymes thatpreferentially perform their primary function at lower or highertemperature as compared to their optimal functional temperature) beforethe array is contacted with the biological sample. In some embodiments,step (e) is performed without bringing the biological sample out ofcontact with the array. In some embodiments, step (e) is performed afterthe biological sample is no longer in contact with the array. In someembodiments, the biological sample is tagged with an analyte captureagent before, contemporaneously with, or after staining and/or imagingof the biological sample. In such cases, significant time (e.g., days,months, or years) can elapse between staining and/or imaging andperforming analysis. In some embodiments, the array is adapted tofacilitate biological analyte migration from the stained and/or imagedbiological sample onto the array (e.g., using any of the materials ormethods described herein). In some embodiments, a biological sample ispermeabilized before being contacted with an array. In some embodiments,the rate of permeabilization is slowed prior to contacting a biologicalsample with an array (e.g., to limit diffusion of analytes away fromtheir original locations in the biological sample). In some embodiments,modulating the rate of permeabilization (e.g., modulating the activityof a permeabilization reagent) can occur by modulating a condition thatthe biological sample is exposed to (e.g., modulating temperature, pH,and/or light). In some embodiments, modulating the rate ofpermeabilization includes use of external stimuli (e.g., smallmolecules, enzymes, and/or activating reagents) to modulate the rate ofpermeabilization. For example, a permeabilization reagent can beprovided to a biological sample prior to contact with an array, whichpermeabilization reagent is inactive until a condition (e.g.,temperature, pH, and/or light) is changed or an external stimulus (e.g.,a small molecule, an enzyme, and/or an activating reagent) is provided.

In some embodiments, provided herein are methods for spatially detectingan analyte (e.g., detecting the location of an analyte, e.g., abiological analyte) from a biological sample (e.g., present in abiological sample such as a tissue section) that include: (a) providinga biological sample on a substrate; (b) staining the biological sampleon the substrate, imaging the stained biological sample, and selectingthe biological sample or subsection of the biological sample (e.g., aregion of interest) to subject to spatial transcriptomic analysis; (c)providing an array comprising one or more pluralities of capture probeson a substrate; (d) contacting the biological sample with the array,thereby allowing a capture probe of the one or more pluralities ofcapture probes to capture the biological analyte of interest; and (e)analyzing the captured biological analyte, thereby spatially detectingthe biological analyte of interest.

(B) Capture Probes

A “capture probe” refers to any molecule capable of capturing (directlyor indirectly) and/or labelling an analyte (e.g., an analyte ofinterest) in a biological sample. In some embodiments, the capture probeis a nucleic acid or a polypeptide. In some embodiments, the captureprobe is a conjugate (e.g., an oligonucleotide-antibody conjugate). Insome embodiments, the capture probe includes a barcode (e.g., a spatialbarcode and/or a unique molecular identifier (UMI)) and a capturedomain.

FIG. 6 is a schematic diagram showing an example of a capture probe, asdescribed herein. As shown, the capture probe 602 is optionally coupledto a feature 601 by a cleavage domain 603, such as a disulfide linker.The capture probe can include functional sequences that are useful forsubsequent processing, such as functional sequence 604, which caninclude a sequencer specific flow cell attachment sequence, e.g., a P5sequence, as well as functional sequence 606, which can includesequencing primer sequences, e.g., a R1 primer binding site. In someembodiments, sequence 604 is a P7 sequence and sequence 606 is a R2primer binding site. A spatial barcode 605 can be included within thecapture probe for use in barcoding the target analyte. The functionalsequences can generally be selected for compatibility with any of avariety of different sequencing systems, e.g., 454 Sequencing, IonTorrent Proton or PGM, Illumina X10, PacBio, Nanopore, etc., and therequirements thereof. In some embodiments, functional sequences can beselected for compatibility with non-commercialized sequencing systems.Examples of such sequencing systems and techniques, for which suitablefunctional sequences can be used, include (but are not limited to) Roche454 sequencing, Ion Torrent Proton or PGM sequencing, Illumina X10sequencing, PacBio SMRT sequencing, and Oxford Nanopore sequencing.Further, in some embodiments, functional sequences can be selected forcompatibility with other sequencing systems, includingnon-commercialized sequencing systems.

In some embodiments, the spatial barcode 605, functional sequences 604(e.g., flow cell attachment sequence) and 606 (e.g., sequencing primersequences) can be common to all of the probes attached to a givenfeature. The spatial barcode can also include a capture domain 607 tofacilitate capture of a target analyte.

(I) Capture Domain

As discussed above, each capture probe includes at least one capturedomain. The “capture domain” can be an oligonucleotide, a polypeptide, asmall molecule, or any combination thereof, that binds specifically to adesired analyte. In some embodiments, a capture domain can be used tocapture or detect a desired analyte.

In some embodiments, the capture domain is a functional nucleic acidsequence configured to interact with one or more analytes, such as oneor more different types of nucleic acids (e.g., RNA molecules and DNAmolecules). In some embodiments, the functional nucleic acid sequencecan include an N-mer sequence (e.g., a random N-mer sequence), whichN-mer sequences are configured to interact with a plurality of DNAmolecules. In some embodiments, the functional sequence can include apoly(T) sequence, which poly(T) sequences are configured to interactwith messenger RNA (mRNA) molecules via the poly(A) tail of an mRNAtranscript. In some embodiments, the functional nucleic acid sequence isthe binding target of a protein (e.g., a transcription factor, a DNAbinding protein, or a RNA binding protein), where the analyte ofinterest is a protein.

Capture probes can include ribonucleotides and/or deoxyribonucleotidesas well as synthetic nucleotide residues that are capable ofparticipating in Watson-Crick type or analogous base pair interactions.In some embodiments, the capture domain is capable of priming a reversetranscription reaction to generate cDNA that is complementary to thecaptured RNA molecules. In some embodiments, the capture domain of thecapture probe can prime a DNA extension (polymerase) reaction togenerate DNA that is complementary to the captured DNA molecules. Insome embodiments, the capture domain can template a ligation reactionbetween the captured DNA molecules and a surface probe that is directlyor indirectly immobilized on the substrate. In some embodiments, thecapture domain can be ligated to one strand of the captured DNAmolecules. For example, SplintR ligase along with RNA or DNA sequences(e.g., degenerate RNA) can be used to ligate a single-stranded DNA orRNA to the capture domain. In some embodiments, ligases withRNA-templated ligase activity, e.g., SplintR ligase, T4 RNA ligase 2 orKOD ligase, can be used to ligate a single-stranded DNA or RNA to thecapture domain. In some embodiments, a capture domain includes a splintoligonucleotide. In some embodiments, a capture domain captures a splintoligonucleotide.

In some embodiments, the capture domain is located at the 3′ end of thecapture probe and includes a free 3′ end that can be extended, e.g., bytemplate dependent polymerization, to form an extended capture probe asdescribed herein. In some embodiments, the capture domain includes anucleotide sequence that is capable of hybridizing to nucleic acid,e.g., RNA or other analyte, present in the cells of the biologicalsample contacted with the array. In some embodiments, the capture domaincan be selected or designed to bind selectively or specifically to atarget nucleic acid. For example, the capture domain can be selected ordesigned to capture mRNA by way of hybridization to the mRNA poly(A)tail. Thus, in some embodiments, the capture domain includes a poly(T)DNA oligonucleotide, e.g., a series of consecutive deoxythymidineresidues linked by phosphodiester bonds, which is capable of hybridizingto the poly(A) tail of mRNA. In some embodiments, the capture domain caninclude nucleotides that are functionally or structurally analogous to apoly(T) tail. For example, a poly(U) oligonucleotide or anoligonucleotide included of deoxythymidine analogues. In someembodiments, the capture domain includes at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, the capturedomain includes at least 25, 30, or 35 nucleotides.

In some embodiments, a capture probe includes a capture domain having asequence that is capable of binding to mRNA and/or genomic DNA. Forexample, the capture probe can include a capture domain that includes anucleic acid sequence (e.g., a poly(T) sequence) capable of binding to apoly(A) tail of an mRNA and/or to a poly(A) homopolymeric sequencepresent in genomic DNA. In some embodiments, a homopolymeric sequence isadded to an mRNA molecule or a genomic DNA molecule using a terminaltransferase enzyme in order to produce an analyte that has a poly(A) orpoly(T) sequence. For example, a poly(A) sequence can be added to ananalyte (e.g., a fragment of genomic DNA) thereby making the analytecapable of capture by a poly(T) capture domain.

In some embodiments, random sequences, e.g., random hexamers or similarsequences, can be used to form all or a part of the capture domain. Forexample, random sequences can be used in conjunction with poly(T) (orpoly(T) analogue) sequences. Thus, where a capture domain includes apoly(T) (or a “poly(T)-like”) oligonucleotide, it can also include arandom oligonucleotide sequence (e.g., “poly(T)-random sequence” probe).This can, for example, be located 5′ or 3′ of the poly(T) sequence,e.g., at the 3′ end of the capture domain. The poly(T)-random sequenceprobe can facilitate the capture of the mRNA poly(A) tail. In someembodiments, the capture domain can be an entirely random sequence. Insome embodiments, degenerate capture domains can be used.

In some embodiments, a pool of two or more capture probes form amixture, where the capture domain of one or more capture probes includesa poly(T) sequence and the capture domain of one or more capture probesincludes random sequences. In some embodiments, a pool of two or morecapture probes form a mixture where the capture domain of one or morecapture probes includes poly(T)-like sequence and the capture domain ofone or more capture probes includes random sequences. In someembodiments, a pool of two or more capture probes form a mixture wherethe capture domain of one or more capture probes includes apoly(T)-random sequences and the capture domain of one or more captureprobes includes random sequences. In some embodiments, probes withdegenerate capture domains can be added to any of the precedingcombinations listed herein. In some embodiments, probes with degeneratecapture domains can be substituted for one of the probes in each of thepairs described herein.

The capture domain can be based on a particular gene sequence orparticular motif sequence or common/conserved sequence, that it isdesigned to capture (i.e., a sequence-specific capture domain). Thus, insome embodiments, the capture domain is capable of binding selectivelyto a desired sub-type or subset of nucleic acid, for example aparticular type of RNA, such as mRNA, rRNA, tRNA, SRP RNA, tmRNA, snRNA,snoRNA, SmY RNA, scaRNA, gRNA, RNase P, RNase MRP, TERC, SL RNA, aRNA,cis-NAT, crRNA, lncRNA, miRNA, piRNA, siRNA, shRNA, tasiRNA, rasiRNA,7SK, eRNA, ncRNA or other types of RNA. In a non-limiting example, thecapture domain can be capable of binding selectively to a desired subsetof ribonucleic acids, for example, microbiome RNA, such as 16S rRNA.

In some embodiments, a capture domain includes an “anchor” or “anchoringsequence”, which is a sequence of nucleotides that is designed to ensurethat the capture domain hybridizes to the intended analyte. In someembodiments, an anchor sequence includes a sequence of nucleotides,including a 1-mer, 2-mer, 3-mer or longer sequence. In some embodiments,the short sequence is random. For example, a capture domain including apoly(T) sequence can be designed to capture an mRNA. In suchembodiments, an anchoring sequence can include a random 3-mer (e.g.,GGG) that helps ensure that the poly(T) capture domain hybridizes to anmRNA. In some embodiments, an anchoring sequence can be VN, N, or NN.Alternatively, the sequence can be designed using a specific sequence ofnucleotides. In some embodiments, the anchor sequence is at the 3′ endof the capture domain. In some embodiments, the anchor sequence is atthe 5′ end of the capture domain.

In some embodiments, capture domains of capture probes are blocked priorto contacting the biological sample with the array, and blocking probesare used when the nucleic acid in the biological sample is modifiedprior to its capture on the array. In some embodiments, the blockingprobe is used to block or modify the free 3′ end of the capture domain.In some embodiments, blocking probes can be hybridized to the captureprobes to mask the free 3′ end of the capture domain, e.g., hairpinprobes, partially double stranded probes, or complementary sequences. Insome embodiments, the free 3′ end of the capture domain can be blockedby chemical modification, e.g., addition of an azidomethyl group as achemically reversible capping moiety such that the capture probes do notinclude a free 3′ end. Blocking or modifying the capture probes,particularly at the free 3′ end of the capture domain, prior tocontacting the biological sample with the array, prevents modificationof the capture probes, e.g., prevents the addition of a poly(A) tail tothe free 3′ end of the capture probes.

Non-limiting examples of 3′ modifications include dideoxy C-3′ (3′-ddC),3′ inverted dT, 3′ C3 spacer, 3′ Amino, and 3′ phosphorylation. In someembodiments, the nucleic acid in the biological sample can be modifiedsuch that it can be captured by the capture domain. For example, anadaptor sequence (including a binding domain capable of binding to thecapture domain of the capture probe) can be added to the end of thenucleic acid, e.g., fragmented genomic DNA. In some embodiments, this isachieved by ligation of the adaptor sequence or extension of the nucleicacid. In some embodiments, an enzyme is used to incorporate additionalnucleotides at the end of the nucleic acid sequence, e.g., a poly(A)tail. In some embodiments, the capture probes can be reversibly maskedor modified such that the capture domain of the capture probe does notinclude a free 3′ end. In some embodiments, the 3′ end is removed,modified, or made inaccessible so that the capture domain is notsusceptible to the process used to modify the nucleic acid of thebiological sample, e.g., ligation or extension.

In some embodiments, the capture domain of the capture probe is modifiedto allow the removal of any modifications of the capture probe thatoccur during modification of the nucleic acid molecules of thebiological sample. In some embodiments, the capture probes can includean additional sequence downstream of the capture domain, e.g., 3′ to thecapture domain, namely a blocking domain.

In some embodiments, the capture domain of the capture probe can be anon-nucleic acid domain. Examples of suitable capture domains that arenot exclusively nucleic-acid based include, but are not limited to,proteins, peptides, aptamers, antigens, antibodies, and molecularanalogs that mimic the functionality of any of the capture domainsdescribed herein.

(II) Cleavage Domain

Each capture probe can optionally include at least one cleavage domain.The cleavage domain represents the portion of the probe that is used toreversibly attach the probe to an array feature, as will be describedfurther herein. Further, one or more segments or regions of the captureprobe can optionally be released from the array feature by cleavage ofthe cleavage domain. As an example, spatial barcodes and/or universalmolecular identifiers (UMIs) can be released by cleavage of the cleavagedomain.

FIG. 7 is a schematic illustrating a cleavable capture probe, whereinthe cleaved capture probe can enter into a non-permeabilized cell andbind to analytes within the sample. The capture probe 701 contains acleavage domain 702, a cell penetrating peptide 703, a reporter molecule704, and a disulfide bond (—S—S—). 705 represents all other parts of acapture probe, for example a spatial barcode and a capture domain.

In some embodiments, the cleavage domain linking the capture probe to afeature is a disulfide bond. A reducing agent can be added to break thedisulfide bonds, resulting in release of the capture probe from thefeature. As another example, heating can also result in degradation ofthe cleavage domain and release of the attached capture probe from thearray feature. In some embodiments, laser radiation is used to heat anddegrade cleavage domains of capture probes at specific locations. Insome embodiments, the cleavage domain is a photo-sensitive chemical bond(e.g., a chemical bond that dissociates when exposed to light such asultraviolet light).

Oligonucleotides with photo-sensitive chemical bonds (e.g.,photo-cleavable linkers) have various advantages. They can be cleavedefficiently and rapidly (e.g., in nanoseconds and milliseconds). In somecases, photo-masks can be used such that only specific regions of thearray are exposed to cleavable stimuli (e.g., exposure to UV light,exposure to light, exposure to heat induced by laser). When aphoto-cleavable linker is used, the cleavable reaction is triggered bylight, and can be highly selective to the linker and consequentlybiorthogonal. Typically, wavelength absorption for the photocleavablelinker is located in the near-UV range of the spectrum. In someembodiments, λ_(max) of the photocleavable linker is from about 300 nmto about 400 nm, or from about 310 nm to about 365 nm. In someembodiments, λmax of the photocleavable linker is about 300 nm, about312 nm, about 325 nm, about 330 nm, about 340 nm, about 345 nm, about355 nm, about 365 nm, or about 400 nm.

Non-limiting examples of a photo-sensitive chemical bond that can beused in a cleavage domain include those described in Leriche et al.Bioorg Med Chem. 2012 Jan 15;20(2):571-82 and U.S. Publication No.2017/0275669, both of which are incorporated by reference herein intheir entireties. For example, linkers that comprise photo-sensitivechemical bonds include 3-amino-3-(2-nitrophenyl)propionic acid (ANP),phenacyl ester derivatives, 8-quinolinyl benzenesulfonate, dicoumarin,6-bromo-7-alkixycoumarin-4-ylmethoxycarbonyl, a bimane-based linker, anda bis-arylhydrazone based linker. In some embodiments, thephoto-sensitive bond is part of a cleavable linker such as anortho-nitrobenzyl (ONB) linker below:

wherein:

-   X is selected from O and NH;-   R¹ is selected from H and C₁₋₃ alkyl;-   R² is selected from H and C₁₋₃ alkoxy;-   n is 1, 2, or 3; and-   a and b each represent either the point of attachment of the linker    to the substrate, or the point of attachment of the linker to the    capture probe.

In some embodiments, at least one spacer is included between thesubstrate and the ortho-nitrobenzyl (ONB) linker, and at least onespacer is included between the ortho-nitrobenzyl (ONB) linker and thecapture probe. In some aspects of these embodiments, the spacercomprises at least one group selected from C1-6 alkylene, C2-6alkenylene, C2-6 alkynylene, C═O, O, S, NH, —(C═O)O—, —(C═O)NH—, —S—S—,ethylene glycol, polyethyleneglycol, propylene glycol, andpolypropyleneglycol, or any combination thereof. In some embodiments, Xis O. In some embodiments, X is NH. In some embodiments, R¹ is H. Insome embodiments, R¹ is C₁₋₃ alkyl. In some embodiments, R¹ is methyl.In some embodiments, R² is H. In some embodiments, R² is C₁₋₃ alkoxy. Insome embodiments, R² is methoxy. In some embodiments, R¹ is H and R² isH. In some embodiments, R¹ is H and R² is methoxy. In some embodiments,R¹ is methyl and R² is H. In some embodiments, R¹ is methyl and R² ismethoxy.

In some embodiments, the photocleavable linker has formula:

In some embodiments, the photocleavable linker has formula:

In some embodiments, the photocleavable linker has formula:

In some embodiments, the photocleavable linker has formula:

In some embodiments, the photocleavable linker has formula:

Without being bound to any particular theory, it is believed thatexcitation of the ortho-nitrobenzyl (ONB) linker leads to Norrish-typehydrogen abstraction in the γ-position, followed by formation of azinicacid, which is highly reactive and rearranges into nitroso compound,resulting in the complete cleavage of the linker, as shown on thefollowing scheme:

In some embodiments, the photocleavable linker is3-amino-3-(2-nitrophenyl)propionic acid (ANP) linker:

wherein X, R², n, a, and b are as described herein for theortho-nitrobenzyl (ONB) linker.

In some embodiments, the photocleavable linker has formula:

In some embodiments, the photocleavable linker is phenacyl ester linker:

wherein a and b are as described herein for the ortho-nitrobenzyl (ONB)linker.

Other examples of photo-sensitive chemical bonds that can be used in acleavage domain include halogenated nucleosides such asbromodeoxyuridine (BrdU). Brdu is an analog of thymidine that can bereadily incorporated into oligonucleotides (e.g., in the cleavage domainof a capture probe), and is sensitive to UVB light (280-320 nm range).Upon exposure to UVB light, a photo-cleavage reaction occurs (e.g., at anucleoside immediately 5′ to the site of Brdu incorporation (Doddridgeet al. Chem. Comm., 1998, 18:1997-1998 and Cook et al. Chemistry andBiology. 1999, 6:451-459)) that results in release of the capture probefrom the feature.

Other examples of cleavage domains include labile chemical bonds suchas, but not limited to, ester linkages (e.g., cleavable with an acid, abase, or hydroxylamine), a vicinal diol linkage (e.g., cleavable viasodium periodate), a Diels-Alder linkage (e.g., cleavable via heat), asulfone linkage (e.g., cleavable via a base), a silyl ether linkage(e.g., cleavable via an acid), a glycosidic linkage (e.g., cleavable viaan amylase), a peptide linkage (e.g., cleavable via a protease), anabasic or apurinic/apyrimidinic (AP) site (e.g., cleavable with analkali or an AP endonuclease), or a phosphodiester linkage (e.g.,cleavable via a nuclease (e.g., DNAase)).

In some embodiments, the cleavage domain includes a sequence that isrecognized by one or more enzymes capable of cleaving a nucleic acidmolecule, e.g., capable of breaking the phosphodiester linkage betweentwo or more nucleotides. A bond can be cleavable via other nucleic acidmolecule targeting enzymes, such as restriction enzymes (e.g.,restriction endonucleases). For example, the cleavage domain can includea restriction endonuclease (restriction enzyme) recognition sequence.Restriction enzymes cut double-stranded or single stranded DNA atspecific recognition nucleotide sequences known as restriction sites. Insome embodiments, a rare-cutting restriction enzyme, e.g., enzymes witha long recognition site (at least 8 base pairs in length), is used toreduce the possibility of cleaving elsewhere in the capture probe.

In some embodiments, the cleavage domain includes a poly(U) sequencewhich can be cleaved by a mixture of Uracil DNA glycosylase (UDG) andthe DNA glycosylase-lyase Endonuclease VIII, commercially known as theUSER™ enzyme. Releasable capture probes can be available for reactiononce released. Thus, for example, an activatable capture probe can beactivated by releasing the capture probes from a feature.

In some embodiments, where the capture probe is attached indirectly to asubstrate, e.g., via a surface probe, the cleavage domain includes oneor more mismatch nucleotides, so that the complementary parts of thesurface probe and the capture probe are not 100% complementary (forexample, the number of mismatched base pairs can be one, two, or threebase pairs). Such a mismatch is recognized, e.g., by the MutY and T7endonuclease I enzymes, which results in cleavage of the nucleic acidmolecule at the position of the mismatch. As described herein a “surfaceprobe” can be any moiety present on the surface of the substrate capableof attaching to an agent (e.g., a capture probe). In some embodiments,the surface probe is an oligonucleotide. In some embodiments, thesurface probe is part of the capture probe.

In some embodiments, where the capture probe is attached (e.g.,immobilized) to a feature indirectly, e.g., via a surface probe, thecleavage domain includes a nickase recognition site or sequence.Nickases are endonucleases which cleave only a single strand of a DNAduplex. Thus, the cleavage domain can include a nickase recognition siteclose to the 5′ end of the surface probe (and/or the 5′ end of thecapture probe) such that cleavage of the surface probe or capture probedestabilizes the duplex between the surface probe and capture probethereby releasing the capture probe) from the feature.

Nickase enzymes can also be used in some embodiments where the captureprobe is attached (e.g., immobilized) to the feature directly. Forexample, the substrate can be contacted with a nucleic acid moleculethat hybridizes to the cleavage domain of the capture probe to provideor reconstitute a nickase recognition site, e.g., a cleavage helperprobe. Thus, contact with a nickase enzyme will result in cleavage ofthe cleavage domain thereby releasing the capture probe from thefeature. Such cleavage helper probes can also be used to provide orreconstitute cleavage recognition sites for other cleavage enzymes,e.g., restriction enzymes.

Some nickases introduce single-stranded nicks only at particular siteson a DNA molecule, by binding to and recognizing a particular nucleotiderecognition sequence. A number of naturally-occurring nickases have beendiscovered, of which at present the sequence recognition properties havebeen determined for at least four. Nickases are described in U.S. Pat.No. 6,867,028, which is incorporated herein by reference in itsentirety. In general, any suitable nickase can be used to bind to acomplementary nickase recognition site of a cleavage domain. Followinguse, the nickase enzyme can be removed from the assay or inactivatedfollowing release of the capture probes to prevent unwanted cleavage ofthe capture probes.

Examples of suitable capture domains that are not exclusivelynucleic-acid based include, but are not limited to, proteins, peptides,aptamers, antigens, antibodies, and molecular analogs that mimic thefunctionality of any of the capture domains described herein.

In some embodiments, a cleavage domain is absent from the capture probe.Examples of substrates with attached capture probes lacking a cleavagedomain are described for example in Macosko et al., (2015) Cell 161,1202-1214, the entire contents of which are incorporated herein byreference.

In some embodiments, the region of the capture probe corresponding tothe cleavage domain can be used for some other function. For example, anadditional region for nucleic acid extension or amplification can beincluded where the cleavage domain would normally be positioned. In suchembodiments, the region can supplement the functional domain or evenexist as an additional functional domain. In some embodiments, thecleavage domain is present but its use is optional.

(III) Functional Domain

Each capture probe can optionally include at least one functionaldomain. Each functional domain typically includes a functionalnucleotide sequence for a downstream analytical step in the overallanalysis procedure.

In some embodiments, the capture probe can include a functional domainfor attachment to a sequencing flow cell, such as, for example, a P5sequence for Illumina® sequencing. In some embodiments, the captureprobe or derivative thereof can include another functional domain, suchas, for example, a P7 sequence for attachment to a sequencing flow cellfor Illumina® sequencing. The functional domains can be selected forcompatibility with a variety of different sequencing systems, e.g., 454Sequencing, Ion Torrent Proton or PGM, Illumina X10, etc., and therequirements thereof.

In some embodiments, the functional domain includes a primer. The primercan include an R1 primer sequence for Illumina® sequencing, and in someembodiments, an R2 primer sequence for Illumina® sequencing. Examples ofsuch capture probes and uses thereof are described in U.S. Pat.Publication Nos. 2014/0378345 and 2015/0376609, the entire contents ofeach of which are incorporated herein by reference.

(IV) Spatial Barcode

As discussed above, the capture probe can include one or more spatialbarcodes (e.g., two or more, three or more, four or more, five or more)spatial barcodes. A “spatial barcode” is a contiguous nucleic acidsegment or two or more non-contiguous nucleic acid segments thatfunction as a label or identifier that conveys or is capable ofconveying spatial information. In some embodiments, a capture probeincludes a spatial barcode that possesses a spatial aspect, where thebarcode is associated with a particular location within an array or aparticular location on a substrate.

A spatial barcode can be part of an analyte, or independent from ananalyte (e.g., part of the capture probe). A spatial barcode can be atag attached to an analyte (e.g., a nucleic acid molecule) or acombination of a tag in addition to an endogenous characteristic of theanalyte (e.g., size of the analyte or end sequence(s)). A spatialbarcode can be unique. In some embodiments where the spatial barcode isunique, the spatial barcode functions both as a spatial barcode and as aunique molecular identifier (UMI), associated with one particularcapture probe.

Spatial barcodes can have a variety of different formats. For example,spatial barcodes can include polynucleotide spatial barcodes; randomnucleic acid and/or amino acid sequences; and synthetic nucleic acidand/or amino acid sequences. In some embodiments, a spatial barcode isattached to an analyte in a reversible or irreversible manner. In someembodiments, a spatial barcode is added to, for example, a fragment of aDNA or RNA sample before, during, and/or after sequencing of the sample.In some embodiments, a spatial barcode allows for identification and/orquantification of individual sequencing-reads. In some embodiments, aspatial barcode is a used as a fluorescent barcode for whichfluorescently labeled oligonucleotide probes hybridize to the spatialbarcode.

In some embodiments, the spatial barcode is a nucleic acid sequence thatdoes not substantially hybridize to analyte nucleic acid molecules in abiological sample. In some embodiments, the spatial barcode has lessthan 80% sequence identity (e.g., less than 70%, 60%, 50%, or less than40% sequence identity) to the nucleic acid sequences across asubstantial part (e.g., 80% or more) of the nucleic acid molecules inthe biological sample.

The spatial barcode sequences can include from about 6 to about 20 ormore nucleotides within the sequence of the capture probes. In someembodiments, the length of a spatial barcode sequence can be about 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer.In some embodiments, the length of a spatial barcode sequence can be atleast about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20nucleotides or longer. In some embodiments, the length of a spatialbarcode sequence is at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 nucleotides or shorter.

These nucleotides can be completely contiguous, e.g., in a singlestretch of adjacent nucleotides, or they can be separated into two ormore separate subsequences that are separated by 1 or more nucleotides.Separated spatial barcode subsequences can be from about 4 to about 16nucleotides in length. In some embodiments, the spatial barcodesubsequence can be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16nucleotides or longer. In some embodiments, the spatial barcodesubsequence can be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16 nucleotides or longer. In some embodiments, the spatial barcodesubsequence can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16 nucleotides or shorter.

For multiple capture probes that are attached to a common array feature,the one or more spatial barcode sequences of the multiple capture probescan include sequences that are the same for all capture probes coupledto the feature, and/or sequences that are different across all captureprobes coupled to the feature.

FIG. 8 is a schematic diagram of an exemplary multiplexedspatially-barcoded feature. In FIG. 8 , the feature 801 can be coupledto spatially-barcoded capture probes, wherein the spatially-barcodedprobes of a particular feature can possess the same spatial barcode, buthave different capture domains designed to associate the spatial barcodeof the feature with more than one target analyte. For example, a featuremay be coupled to four different types of spatially-barcoded captureprobes, each type of spatially-barcoded capture probe possessing thespatial barcode 802. One type of capture probe associated with thefeature includes the spatial barcode 802 in combination with a poly(T)capture domain 803, designed to capture mRNA target analytes. A secondtype of capture probe associated with the feature includes the spatialbarcode 802 in combination with a random N-mer capture domain 804 forgDNA analysis. A third type of capture probe associated with the featureincludes the spatial barcode 802 in combination with a capture domaincomplementary to the capture domain on an analyte capture agent captureagent barcode domain 805. A fourth type of capture probe associated withthe feature includes the spatial barcode 802 in combination with acapture probe that can specifically bind a nucleic acid molecule 806that can function in a CRISPR assay (e.g., CRISPR/Cas9). While only fourdifferent capture probe-barcoded constructs are shown in FIG. 8 ,capture-probe barcoded constructs can be tailored for analyses of anygiven analyte associated with a nucleic acid and capable of binding withsuch a construct. For example, the schemes shown in FIG. 8 can also beused for concurrent analysis of other analytes disclosed herein,including, but not limited to: (a) mRNA, a lineage tracing construct,cell surface or intracellular proteins and metabolites, and gDNA; (b)mRNA, accessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq)cell surface or intracellular proteins and metabolites, and aperturbation agent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc fingernuclease, and/or antisense oligonucleotide as described herein); (c)mRNA, cell surface or intracellular proteins and/or metabolites, abarcoded labelling agent (e.g., the MHC multimers described herein), anda V(D)J sequence of an immune cell receptor (e.g., T-cell receptor). Insome embodiments, a perturbation agent can be a small molecule, anantibody, a drug, an aptamer, a miRNA, a physical environmental (e.g.,temperature change), or any other known perturbation agents.

Capture probes attached to a single array feature can include identical(or common) spatial barcode sequences, different spatial barcodesequences, or a combination of both. Capture probes attached to afeature can include multiple sets of capture probes. Capture probes of agiven set can include identical spatial barcode sequences. The identicalspatial barcode sequences can be different from spatial barcodesequences of capture probes of another set.

The plurality of capture probes can include spatial barcode sequences(e.g., nucleic acid barcode sequences) that are associated with specificlocations on a spatial array. For example, a first plurality of captureprobes can be associated with a first region, based on a spatial barcodesequence common to the capture probes within the first region, and asecond plurality of capture probes can be associated with a secondregion, based on a spatial barcode sequence common to the capture probeswithin the second region. The second region may or may not be associatedwith the first region. Additional pluralities of capture probes can beassociated with spatial barcode sequences common to the capture probeswithin other regions. In some embodiments, the spatial barcode sequencescan be the same across a plurality of capture probe molecules.

In some embodiments, multiple different spatial barcodes areincorporated into a single arrayed capture probe. For example, a mixedbut known set of spatial barcode sequences can provide a strongeraddress or attribution of the spatial barcodes to a given spot orlocation, by providing duplicate or independent confirmation of theidentity of the location. In some embodiments, the multiple spatialbarcodes represent increasing specificity of the location of theparticular array point.

(V) Unique Molecular Identifier

The capture probe can include one or more (e.g., two or more, three ormore, four or more, five or more) Unique Molecular Identifiers (UMIs). Aunique molecular identifier is a contiguous nucleic acid segment or twoor more non-contiguous nucleic acid segments that function as a label oridentifier for a particular analyte, or for a capture probe that binds aparticular analyte (e.g., via the capture domain).

A UMI can be unique. A UMI can include one or more specificpolynucleotides sequences, one or more random nucleic acid and/or aminoacid sequences, and/or one or more synthetic nucleic acid and/or aminoacid sequences.

In some embodiments, the UMI is a nucleic acid sequence that does notsubstantially hybridize to analyte nucleic acid molecules in abiological sample. In some embodiments, the UMI has less than 80%sequence identity (e.g., less than 70%, 60%, 50%, or less than 40%sequence identity) to the nucleic acid sequences across a substantialpart (e.g., 80% or more) of the nucleic acid molecules in the biologicalsample.

The UMI can include from about 6 to about 20 or more nucleotides withinthe sequence of the capture probes. In some embodiments, the length of aUMI sequence can be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 nucleotides or longer. In some embodiments, the length of aUMI sequence can be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 nucleotides or longer. In some embodiments, thelength of a UMI sequence is at most about 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 nucleotides or shorter.

These nucleotides can be completely contiguous, i.e., in a singlestretch of adjacent nucleotides, or they can be separated into two ormore separate subsequences that are separated by 1 or more nucleotides.Separated UMI subsequences can be from about 4 to about 16 nucleotidesin length. In some embodiments, the UMI subsequence can be about 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In someembodiments, the UMI subsequence can be at least about 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments,the UMI subsequence can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16 nucleotides or shorter.

In some embodiments, a UMI is attached to an analyte in a reversible orirreversible manner. In some embodiments, a UMI is added to, forexample, a fragment of a DNA or RNA sample before, during, and/or aftersequencing of the analyte. In some embodiments, a UMI allows foridentification and/or quantification of individual sequencing-reads. Insome embodiments, a UMI is a used as a fluorescent barcode for whichfluorescently labeled oligonucleotide probes hybridize to the UMI.

(VI) Other Aspects of Capture Probes

For capture probes that are attached to an array feature, an individualarray feature can include one or more capture probes. In someembodiments, an individual array feature includes hundreds or thousandsof capture probes. In some embodiments, the capture probes areassociated with a particular individual feature, where the individualfeature contains a capture probe including a spatial barcode unique to adefined region or location on the array.

In some embodiments, a particular feature can contain capture probesincluding more than one spatial barcode (e.g., one capture probe at aparticular feature can include a spatial barcode that is different thanthe spatial barcode included in another capture probe at the sameparticular feature, while both capture probes include a second, commonspatial barcode), where each spatial barcode corresponds to a particulardefined region or location on the array. For example, multiple spatialbarcode sequences associated with one particular feature on an array canprovide a stronger address or attribution to a given location byproviding duplicate or independent confirmation of the location. In someembodiments, the multiple spatial barcodes represent increasingspecificity of the location of the particular array point. In anon-limiting example, a particular array point can be coded with twodifferent spatial barcodes, where each spatial barcode identifies aparticular defined region within the array, and an array pointpossessing both spatial barcodes identifies the sub-region where twodefined regions overlap, e.g., such as the overlapping portion of a Venndiagram.

In another non-limiting example, a particular array point can be codedwith three different spatial barcodes, where the first spatial barcodeidentifies a first region within the array, the second spatial barcodeidentifies a second region, where the second region is a subregionentirely within the first region, and the third spatial barcodeidentifies a third region, where the third region is a subregionentirely within the first and second subregions.

In some embodiments, capture probes attached to array features arereleased from the array features for sequencing. Alternatively, in someembodiments, capture probes remain attached to the array features, andthe probes are sequenced while remaining attached to the array features(e.g., via in situ sequencing). Further aspects of the sequencing ofcapture probes are described in subsequent sections of this disclosure.

In some embodiments, an array feature can include different types ofcapture probes attached to the feature. For example, the array featurecan include a first type of capture probe with a capture domain designedto bind to one type of analyte, and a second type of capture probe witha capture domain designed to bind to a second type of analyte. Ingeneral, array features can include one or more (e.g., two or more,three or more, four or more, five or more, six or more, eight or more,ten or more, 12 or more, 15 or more, 20 or more, 30 or more, 50 or more)different types of capture probes attached to a single array feature.

In some embodiments, the capture probe is nucleic acid. In someembodiments, the capture probe is attached to the array feature via its5′ end. In some embodiments, the capture probe includes from the 5′ to3′ end: one or more barcodes (e.g., a spatial barcode and/or a UMI) andone or more capture domains. In some embodiments, the capture probeincludes from the 5′ to 3′ end: one barcode (e.g., a spatial barcode ora UMI) and one capture domain. In some embodiments, the capture probeincludes from the 5′ to 3′ end: a cleavage domain, a functional domain,one or more barcodes (e.g., a spatial barcode and/or a UMI), and acapture domain. In some embodiments, the capture probe includes from the5′ to 3′ end: a cleavage domain, a functional domain, one or morebarcodes (e.g., a spatial barcode and/or a UMI), a second functionaldomain, and a capture domain. In some embodiments, the capture probeincludes from the 5′ to 3′ end: a cleavage domain, a functional domain,a spatial barcode, a UMI, and a capture domain. In some embodiments, thecapture probe does not include a spatial barcode. In some embodiments,the capture probe does not include a UMI. In some embodiments, thecapture probe includes a sequence for initiating a sequencing reaction.

In some embodiments, the capture probe is immobilized on a feature viaits 3′ end. In some embodiments, the capture probe includes from the 3′to 5′ end: one or more barcodes (e.g., a spatial barcode and/or a UMI)and one or more capture domains. In some embodiments, the capture probeincludes from the 3′ to 5′ end: one barcode (e.g., a spatial barcode ora UMI) and one capture domain. In some embodiments, the capture probeincludes from the 3′ to 5′ end: a cleavage domain, a functional domain,one or more barcodes (e.g., a spatial barcode and/or a UMI), and acapture domain. In some embodiments, the capture probe includes from the3′ to 5′ end: a cleavage domain, a functional domain, a spatial barcode,a UMI, and a capture domain.

In some embodiments, a capture probe includes an in situ synthesizedoligonucleotide. The in situ synthesized oligonucleotide can be attachedto a substrate, or to a feature on a substrate. In some embodiments, thein situ synthesized oligonucleotide includes one or more constantsequences, one or more of which serves as a priming sequence (e.g., aprimer for amplifying target nucleic acids). The in situ synthesizedoligonucleotide can, for example, include a constant sequence at the 3′end that is attached to a substrate, or attached to a feature on asubstrate. Additionally or alternatively, the in situ synthesizedoligonucleotide can include a constant sequence at the free 5′ end. Insome embodiments, the one or more constant sequences can be a cleavablesequence. In some embodiments, the in situ synthesized oligonucleotideincludes a barcode sequence, e.g., a variable barcode sequence. Thebarcode can be any of the barcodes described herein. The length of thebarcode can be approximately 8 to 16 nucleotides (e.g., 8, 9, 10, 11,12, 13, 14, 15, or 16 nucleotides). The length of the in situsynthesized oligonucleotide can be less than 100 nucleotides (e.g., lessthan 90, 80, 75, 70, 60, 50, 45, 40, 35, 30, 25 or 20 nucleotides). Insome instances, the length of the in situ synthesized oligonucleotide isabout 20 to about 40 nucleotides. Exemplary in situ synthesizedoligonucleotides are produced by Affymetrix. In some embodiments, the insitu synthesized oligonucleotide is attached to a feature of an array.

Additional oligonucleotides can be ligated to an in situ synthesizedoligonucleotide to generate a capture probe. For example, a primercomplementary to a portion of the in situ synthesized oligonucleotide(e.g., a constant sequence in the oligonucleotide) can be used tohybridize an additional oligonucleotide and extend (using the in situsynthesized oligonucleotide as a template e.g., a primer extensionreaction) to form a double stranded oligonucleotide and to furthercreate a 3′ overhang. In some embodiments, the 3′ overhang can becreated by template-independent ligases (e.g., terminal deoxynucleotidyltransferase (TdT) or poly(A) polymerase). An additional oligonucleotidecomprising one or more capture domains can be ligated to the 3′ overhangusing a suitable enzyme (e.g., a ligase) and a splint oligonucleotide,to generate a capture probe. Thus, in some embodiments, a capture probeis a product of two or more oligonucleotide sequences, (e.g., the insitu synthesized oligonucleotide and the additional oligonucleotide)that are ligated together. In some embodiments, one of theoligonucleotide sequences is an in situ synthesized oligonucleotide.

In some embodiments, the capture probe can be prepared using a splintoligonucleotide (e.g., any of the splint oligonucleotides describedherein). Two or more oligonucleotides can be ligated together using asplint oligonucleotide and any variety of ligases known in the art ordescribed herein (e.g., SplintR ligase).

One of the oligonucleotides can include, for example, a constantsequence (e.g., a sequence complementary to a portion of a splintoligonucleotide), a degenerate sequence, and/or a capture domain (e.g.,as described herein). One of the oligonucleotides can also include asequence compatible for ligating or hybridizing to an analyte ofinterest in the biological sample. An analyte of interest (e.g., anmRNA) can also be used as a splint oligonucleotide to ligate furtheroligonucleotides onto the capture probe. In some embodiments, thecapture probe is generated by having an enzyme add polynucleotides atthe end of an oligonucleotide sequence. The capture probe can include adegenerate sequence, which can function as a unique molecularidentifier.

A degenerate sequence, which is a sequence in which some positions of anucleotide sequence contain a number of possible bases. A degeneratesequence can be a degenerate nucleotide sequence including about or atleast 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 45, or 50 nucleotides. In some embodiments, a nucleotidesequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or moredegenerate positions within the nucleotide sequence. In someembodiments, the degenerate sequence is used as a UMI.

In some embodiments, a capture probe includes a restriction endonucleaserecognition sequence or a sequence of nucleotides cleavable by specificenzyme activities. For example, uracil sequences can be enzymaticallycleaved from a nucleotide sequence using uracil DNA glycosylase (UDG) orUracil Specific Excision Reagent (USER). As another example, othermodified bases (e.g., modified by methylation) can be recognized andcleaved by specific endonucleases. The capture probes can be subjectedto an enzymatic cleavage, which removes the blocking domain and any ofthe additional nucleotides that are added to the 3′ end of the captureprobe during the modification process. Removal of the blocking domainreveals and/or restores the free 3′ end of the capture domain of thecapture probe. In some embodiments, additional nucleotides can beremoved to reveal and/or restore the 3′ end of the capture domain of thecapture probe.

In some embodiments, a blocking domain can be incorporated into thecapture probe when it is synthesized, or after its synthesis. Theterminal nucleotide of the capture domain is a reversible terminatornucleotide (e.g., 3′—O—blocked reversible terminator and 3′-unblockedreversible terminator), and can be included in the capture probe duringor after probe synthesis.

(VII) Extended Capture Probes

An “extended capture probe” is a capture probe with an enlarged nucleicacid sequence. For example, where the capture probe includes nucleicacid, an “extended 3′ end” indicates that further nucleotides were addedto the most 3′ nucleotide of the capture probe to extend the length ofthe capture probe, for example, by standard polymerization reactionsutilized to extend nucleic acid molecules including templatedpolymerization catalyzed by a polymerase (e.g., a DNA polymerase orreverse transcriptase).

In some embodiments, extending the capture probe includes generatingcDNA from the captured (hybridized) RNA. This process involves synthesisof a complementary strand of the hybridized nucleic acid, e.g.,generating cDNA based on the captured RNA template (the RNA hybridizedto the capture domain of the capture probe). Thus, in an initial step ofextending the capture probe, e.g., the cDNA generation, the captured(hybridized) nucleic acid, e.g., RNA, acts as a template for theextension, e.g., reverse transcription, step.

In some embodiments, the capture probe is extended using reversetranscription. For example, reverse transcription includes synthesizingcDNA (complementary or copy DNA) from RNA, e.g., (messenger RNA), usinga reverse transcriptase. In some embodiments, reverse transcription isperformed while the tissue is still in place, generating an analytelibrary, where the analyte library includes the spatial barcodes fromthe adjacent capture probes. In some embodiments, the capture probe isextended using one or more DNA polymerases.

In some embodiments, the capture domain of the capture probe includes aprimer for producing the complementary strand of the nucleic acidhybridized to the capture probe, e.g., a primer for DNA polymeraseand/or reverse transcription. The nucleic acid, e.g., DNA and/or cDNA,molecules generated by the extension reaction incorporate the sequenceof the capture probe. The extension of the capture probe, e.g., a DNApolymerase and/or reverse transcription reaction, can be performed usinga variety of suitable enzymes and protocols.

In some embodiments, a full-length DNA, e.g., cDNA, molecule isgenerated. In some embodiments, a “full-length” DNA molecule refers tothe whole of the captured nucleic acid molecule. However, if the nucleicacid, e.g., RNA, was partially degraded in the tissue sample, then thecaptured nucleic acid molecules will not be the same length as theinitial RNA in the tissue sample. In some embodiments, the 3′ end of theextended probes, e.g., first strand cDNA molecules, is modified. Forexample, a linker or adaptor can be ligated to the 3′ end of theextended probes. This can be achieved using single stranded ligationenzymes such as T4 RNA ligase or Circligase™ (available from EpicentreBiotechnologies, Madison, WI). In some embodiments, template switchingoligonucleotides are used to extend cDNA in order to generate afull-length cDNA (or as close to a full-length cDNA as possible). Insome embodiments, a second strand synthesis helper probe (a partiallydouble stranded DNA molecule capable of hybridizing to the 3′ end of theextended capture probe), can be ligated to the 3′ end of the extendedprobe, e.g., first strand cDNA, molecule using a double strandedligation enzyme such as T4 DNA ligase. Other enzymes appropriate for theligation step are known in the art and include, e.g., Tth DNA ligase,Taq DNA ligase, Thermococcus sp. (strain 9°N) DNA ligase (9°N™ DNAligase, New England Biolabs), Ampligase™ (available from EpicentreBiotechnologies, Madison, WI), and SplintR (available from New EnglandBiolabs, Ipswich, MA). In some embodiments, a polynucleotide tail, e.g.,a poly(A) tail, is incorporated at the 3′ end of the extended probemolecules. In some embodiments, the polynucleotide tail is incorporatedusing a terminal transferase active enzyme.

In some embodiments, double-stranded extended capture probes are treatedto remove any unextended capture probes prior to amplification and/oranalysis, e.g., sequence analysis. This can be achieved by a variety ofmethods, e.g., using an enzyme to degrade the unextended probes, such asan exonuclease enzyme, or purification columns.

In some embodiments, extended capture probes are amplified to yieldquantities that are sufficient for analysis, e.g., via DNA sequencing.In some embodiments, the first strand of the extended capture probes(e.g., DNA and/or cDNA molecules) acts as a template for theamplification reaction (e.g., a polymerase chain reaction).

In some embodiments, the amplification reaction incorporates an affinitygroup onto the extended capture probe (e.g., RNA-cDNA hybrid) using aprimer including the affinity group. In some embodiments, the primerincludes an affinity group and the extended capture probes includes theaffinity group. The affinity group can correspond to any of the affinitygroups described previously.

In some embodiments, the extended capture probes including the affinitygroup can be coupled to an array feature specific for the affinitygroup. In some embodiments, the substrate can include an antibody orantibody fragment. In some embodiments, the array feature includesavidin or streptavidin and the affinity group includes biotin. In someembodiments, the array feature includes maltose and the affinity groupincludes maltose-binding protein. In some embodiments, the array featureincludes maltose-binding protein and the affinity group includesmaltose. In some embodiments, amplifying the extended capture probes canfunction to release the extended probes from the array feature, insofaras copies of the extended probes are not attached to the array feature.

In some embodiments, the extended capture probe or complement oramplicon thereof is released from an array feature. The step ofreleasing the extended capture probe or complement or amplicon thereoffrom an array feature can be achieved in a number of ways. In someembodiments, an extended capture probe or a complement thereof isreleased from the feature by nucleic acid cleavage and/or bydenaturation (e.g., by heating to denature a double-stranded molecule).

In some embodiments, the extended capture probe or complement oramplicon thereof is released from the array feature by physical means.For example, methods for inducing physical release include denaturingdouble stranded nucleic acid molecules. Another method for releasing theextended capture probes is to use a solution that interferes with thehydrogen bonds of the double stranded molecules. In some embodiments,the extended capture probe is released by applying heated water such aswater or buffer of at least 85° C., e.g., at least 90, 91, 92, 93, 94,95, 96, 97, 98, or 99° C. In some embodiments, a solution includingsalts, surfactants, etc. that can further destabilize the interactionbetween the nucleic acid molecules is added to release the extendedcapture probe from the array feature. In some embodiments, a formamidesolution can be used to destabilize the interaction between nucleic acidmolecules to release the extended capture probe from the array feature.

(VIII) Analyte Capture Agents

This disclosure also provides methods and materials for using analytecapture agents for spatial profiling of biological analytes (e.g., mRNA,genomic DNA, accessible chromatin, and cell surface or intracellularproteins and/or metabolites). As used herein, an “analyte capture agent”(also referred to previously at times as a “cell labelling” agent”)refers to an agent that interacts with an analyte (e.g., an analyte in asample) and with a capture probe (e.g., a capture probe attached to asubstrate) to identify the analyte. In some embodiments, the analytecapture agent includes an analyte binding moiety and a capture agentbarcode domain.

FIG. 9 is a schematic diagram of an exemplary analyte capture agent 902comprised of an analyte binding moiety 904 and a capture agent barcodedomain 908. An analyte binding moiety 904 is a molecule capable ofbinding to an analyte 906 and interacting with a spatially-barcodedcapture probe. The analyte binding moiety can bind to the analyte 906with high affinity and/or with high specificity. The analyte captureagent can include a capture agent barcode domain 908, a nucleotidesequence (e.g., an oligonucleotide), which can hybridize to at least aportion or an entirety of a capture domain of a capture probe. Theanalyte binding moiety 904 can include a polypeptide and/or an aptamer(e.g., an oligonucleotide or peptide molecule that binds to a specifictarget analyte). The analyte binding moiety 904 can include an antibodyor antibody fragment (e.g., an antigen-binding fragment).

As used herein, the term “analyte binding moiety” refers to a moleculeor moiety capable of binding to a macromolecular constituent (e.g., ananalyte, e.g., a biological analyte). In some embodiments of any of thespatial profiling methods described herein, the analyte binding moietyof the analyte capture agent that binds to a biological analyte caninclude, but is not limited to, an antibody, or an epitope bindingfragment thereof, a cell surface receptor binding molecule, a receptorligand, a small molecule, a bi-specific antibody, a bi-specific T-cellengager, a T-cell receptor engager, a B-cell receptor engager, apro-body, an aptamer, a monobody, an affimer, a darpin, and a proteinscaffold, or any combination thereof. The analyte binding moiety canbind to the macromolecular constituent (e.g., analyte) with highaffinity and/or with high specificity. The analyte binding moiety caninclude a nucleotide sequence (e.g., an oligonucleotide), which cancorrespond to at least a portion or an entirety of the analyte bindingmoiety. The analyte binding moiety can include a polypeptide and/or anaptamer (e.g., a polypeptide and/or an aptamer that binds to a specifictarget molecule, e.g., an analyte). The analyte binding moiety caninclude an antibody or antibody fragment (e.g., an antigen-bindingfragment) that binds to a specific analyte (e.g., a polypeptide).

In some embodiments, an analyte binding moiety of an analyte captureagent includes one or more antibodies or antigen binding fragmentsthereof. The antibodies or antigen binding fragments including theanalyte binding moiety can specifically bind to a target analyte. Insome embodiments, the analyte is a protein (e.g., a protein on a surfaceof the biological sample (e.g., a cell) or an intracellular protein). Insome embodiments, a plurality of analyte capture agents comprising aplurality of analyte binding moieties bind a plurality of analytespresent in a biological sample. In some embodiments, the plurality ofanalytes includes a single species of analyte (e.g., a single species ofpolypeptide). In some embodiments in which the plurality of analytesincludes a single species of analyte, the analyte binding moieties ofthe plurality of analyte capture agents are the same. In someembodiments in which the plurality of analytes includes a single speciesof analyte, the analyte binding moieties of the plurality of analytecapture agents are the different (e.g., members of the plurality ofanalyte capture agents can have two or more species of analyte bindingmoieties, wherein each of the two or more species of analyte bindingmoieties binds a single species of analyte, e.g., at different bindingsites). In some embodiments, the plurality of analytes includes multipledifferent species of analyte (e.g., multiple different species ofpolypeptides).

An analyte capture agent can include an analyte binding moiety. Theanalyte binding moiety can be an antibody. Exemplary, non-limitingantibodies that can be used as analyte binding moieties in an analytecapture agent or that can be used in the IHC/IF applications disclosedherein include any of the following including variations thereof:A-ACT,, A-AT, ACTH, Actin-Muscle-specific, Actin-Smooth Muscle (SMA),AE1, AE1/AE3, AE3, AFP, AKT Phosphate, ALK-1, Amyloid A, AndrogenReceptor, Annexin A1, B72.3, BCA-225, BCL-1 (Cyclin D1), BCL-1/CD20,BCL-2, BCL-2/BCL-6, BCL-6, Ber—EP4, Beta-amyloid, Beta-catenin, BG8(Lewis Y), BOB-1, CA 19.9, CA 125, CAIX, Calcitonin, Caldesmon,Calponin, Calretinin, CAM 5.2, CAM 5.2/AE1, CD1a, CD2, CD3 (M), CD3 (P),CD3/CD20, CD4, CD5, CD7, CD8, CD10, CD14, CD15, CD20, CD21, CD22, CD 23,CD25, CD30, CD31, CD33, CD34, CD35, CD43, CD45 (LCA), CD45RA, CD56,CD57, CD61, CD68, CD71, CD74, CD79a, CD99, CD117 (c-KIT), CD123, CD138,CD163, CDX-2, CDX-2/CK-7, CEA (M), CEA (P), Chromogranin A,Chymotrypsin, CK-5, CK-5/6, CK-7, CK-7/TTF-1, CK-14, CK-17, CK-18,CK-19, CK-20, CK-HMW, CK-LMW, CMV-IH, COLL-IV, COX-2, D2-40, DBA44,Desmin, DOG1, EBER-ISH, EBV (LMP1), E-Cadherin, EGFR, EMA, ER, ERCC1,Factor VIII (vWF), Factor XIIIa, Fascin, FLI-1, FHS, Galectin-3,Gastrin, GCDFP-15, GFAP, Glucagon, Glycophorin A, Glypican-3, GranzymeB, Growth Hormone (GH), GST, HAM 56, HMBE-1, HBP, HCAg, HCG, HemoglobinA, HEP B CORE (HBcAg), HEP B SURF, (HBsAg), HepPar1, HER2, Herpes I,Herpes II, HHV-8, HLA-DR, HMB 45, HPL, HPV-IHC, HPV (6/11)-ISH, HPV(16/18)-ISH, HPV (31/33)-ISH, HPV WSS-ISH, HPV High-ISH, HPV Low-ISH,HPV High & Low-ISH, IgA, IgD, IgG, IgG4, IgM, Inhibin, Insulin, JCVirus-ISH, Kappa-ISH, KER PAN, Ki-67, Lambda-IHC, Lambda-ISH, LH,Lipase, Lysozyme (MURA), Mammaglobin, MART-1, MBP, M-Cell Tryptase,MEL-5, Melan-A,, Melan-A/Ki-67, Mesothelin, MiTF, MLH-1, MOC-31, MPO,MSH-2, MSH-6, MUC1, MUC2, MUC4, MUC5AC, MUM-1, MYO D1, Myogenin,Myoglobin, Myoin Heavy Chain, Napsin A, NB84a, NEW-N, NF, NK1—C3, NPM,NSE, OCT-2, OCT-3/4, OSCAR, p16, p21, p27/Kip1, p53, p57, p63, p120,P504S, Pan Melanoma, PANC.POLY, Parvovirus B19, PAX-2, PAX-5,PAX-5/CD43, PAX=5/CD5, PAX-8, PC, PD1, Perforin, PGP 9.5, PLAP, PMS-2,PR, Prolactin, PSA, PSAP, PSMA, PTEN, PTH, PTS, RB, RCC, S6, S100,Serotonin, Somatostatin, Surfactant (SP-A), Synaptophysin, Synuclein,TAU, TCL-1, TCR beta, TdT, Thrombomodulin, Thyroglobulin, TIA-1, TOXO,TRAP, TriView™ breast, TriView™ prostate, Trypsin, TS, TSH, TTF-1,Tyrosinase, Ubiqutin, Uroplakin, VEGF, Villin, Vimentin (VIM), VIP, VZV,WT1 (M) N-Terminus, WT1 (P) C-Terminus, ZAP-70.

Further, exemplary, non-limiting antibodies that can be used as analytebinding moieties in an analyte capture agent or that can be used in theIHC/IF applications disclosed herein include any of the followingantibodies (and variations thereof) to: cell surface proteins,intracellular proteins, kinases (e.g., AGC kinase family (e.g., AKT1,AKT2, PDK1, Protein Kinase C, ROCK1, ROCK2, SGK3), CAMK kinase family(e.g., AMPK1, AMPK2, CAMK, Chk1, Chk2, Zip), CK1 kinase family, TKkinase family (e.g., Abl2, AXL, CD167, CD246/ALK, c-Met, CSK, c-Src,EGFR, ErbB2 (HER2/neu), ErbB3, ErbB4, FAK, Fyn, LCK, Lyn, PKT7, Syk,Zap70), STE kinase family (e.g., ASK1, MAPK, MEK1, MEK2, MEK3 MEK4,MEK5, PAK1, PAK2, PAK4, PAK6), CMGC kinase family (e.g., Cdk2, Cdk4,Cdk5, Cdk6, Cdk7, Cdk9, Erk1, GSK3, Jnk/MAPK8, Jnk2/MAPK9, JNK3/MAPK10,p38/MAPK), and TKL kinase family (e.g., ALK1, ILK1, IRAK1, IRAK2, IRAK3,IRAK4, LIMK1, LIMK2, M3K11, RAF1, RIP1, RIP3, VEGFR1, VEGFR2, VEGFR3),Aurora A kinase, Aurora B kinase, IKK, Nemo-like kinase, PINK, PLK3,ULK2, WEE1, transcription factors (e.g., FOXP3, ATF3, BACH1, EGR, ELF3,FOXA1, FOXA2, FOX01, GATA), growth factor receptors, tumor suppressors(e.g., anti-p53, anti-BLM, anti-Cdk2, anti-Chk2, anti-BRCA-1, anti-NBS1,anti-BRCA-2, anti-WRN, anti-PTEN, anti-WT1, anti-p38).

In some embodiments, analyte capture agents are capable of binding toanalytes present inside a cell. In some embodiments, analyte captureagents are capable of binding to cell surface analytes that can include,without limitation, a receptor, an antigen, a surface protein, atransmembrane protein, a cluster of differentiation protein, a proteinchannel, a protein pump, a carrier protein, a phospholipid, aglycoprotein, a glycolipid, a cell-cell interaction protein complex, anantigen-presenting complex, a major histocompatibility complex, anengineered T-cell receptor, a T-cell receptor, a B-cell receptor, achimeric antigen receptor, an extracellular matrix protein, aposttranslational modification (e.g., phosphorylation, glycosylation,ubiquitination, nitrosylation, methylation, acetylation or lipidation)state of a cell surface protein, a gap junction, and an adherensjunction. In some embodiments, the analyte capture agents are capable ofbinding to cell surface analytes that are post-translationally modified.In such embodiments, analyte capture agents can be specific for cellsurface analytes based on a given state of posttranslationalmodification (e.g., phosphorylation, glycosylation, ubiquitination,nitrosylation, methylation, acetylation or lipidation), such that a cellsurface analyte profile can include posttranslational modificationinformation of one or more analytes.

In some embodiments, the analyte capture agent includes a capture agentbarcode domain that is conjugated or otherwise attached to the analytebinding moiety. In some embodiments, the capture agent barcode domain iscovalently-linked to the analyte binding moiety. In some embodiments, acapture agent barcode domain is a nucleic acid sequence. In someembodiments, a capture agent barcode domain includes an analyte bindingmoiety barcode and an analyte capture sequence.

As used herein, the term “analyte binding moiety barcode” refers to abarcode that is associated with or otherwise identifies the analytebinding moiety. In some embodiments, by identifying an analyte bindingmoiety and its associated analyte binding moiety barcode, the analyte towhich the analyte binding moiety binds can also be identified. Ananalyte binding moiety barcode can be a nucleic acid sequence of a givenlength and/or sequence that is associated with the analyte bindingmoiety. An analyte binding moiety barcode can generally include any ofthe variety of aspects of barcodes described herein. For example, ananalyte capture agent that is specific to one type of analyte can havecoupled thereto a first capture agent barcode domain (e.g., thatincludes a first analyte binding moiety barcode), while an analytecapture agent that is specific to a different analyte can have adifferent capture agent barcode domain (e.g., that includes a secondbarcode analyte binding moiety barcode) coupled thereto. In someaspects, such a capture agent barcode domain can include an analytebinding moiety barcode that permits identification of the analytebinding moiety to which the capture agent barcode domain is coupled. Theselection of the capture agent barcode domain can allow significantdiversity in terms of sequence, while also being readily attachable tomost analyte binding moieties (e.g., antibodies or aptamers) as well asbeing readily detected, (e.g., using sequencing or array technologies).

In some embodiments, the capture agent barcode domain of an analytecapture agent includes an analyte capture sequence. As used herein, theterm “analyte capture sequence” refers to a region or moiety configuredto hybridize to, bind to, couple to, or otherwise interact with acapture domain of a capture probe. In some embodiments, an analytecapture sequence includes a nucleic acid sequence that is complementaryto or substantially complementary to the capture domain of a captureprobe such that the analyte capture sequence hybridizes to the capturedomain of the capture probe. In some embodiments, an analyte capturesequence comprises a poly(A) nucleic acid sequence that hybridizes to acapture domain that comprises a poly(T) nucleic acid sequence. In someembodiments, an analyte capture sequence comprises a poly(T) nucleicacid sequence that hybridizes to a capture domain that comprises apoly(A) nucleic acid sequence. In some embodiments, an analyte capturesequence comprises a non-homopolymeric nucleic acid sequence thathybridizes to a capture domain that comprises a non-homopolymericnucleic acid sequence that is complementary (or substantiallycomplementary) to the non-homopolymeric nucleic acid sequence of theanalyte capture region.

In some embodiments of any of the spatial analysis methods describedherein that employ an analyte capture agent, the capture agent barcodedomain can be directly coupled to the analyte binding moiety, or theycan be attached to a bead, molecular lattice, e.g., a linear, globular,cross-slinked, or other polymer, or other framework that is attached orotherwise associated with the analyte binding moiety, which allowsattachment of multiple capture agent barcode domains to a single analytebinding moiety. Attachment (coupling) of the capture agent barcodedomains to the analyte binding moieties can be achieved through any of avariety of direct or indirect, covalent or non-covalent associations orattachments. For example, in the case of a capture agent barcode domaincoupled to an analyte binding moiety that includes an antibody orantigen-binding fragment, such capture agent barcode domains can becovalently attached to a portion of the antibody or antigen-bindingfragment using chemical conjugation techniques (e.g., Lightning-Link®antibody labelling kits available from Innova Biosciences). In someembodiments, a capture agent barcode domain can be coupled to anantibody or antigen-binding fragment using non-covalent attachmentmechanisms (e.g., using biotinylated antibodies and oligonucleotides orbeads that include one or more biotinylated linker(s), coupled tooligonucleotides with an avidin or streptavidin linker.) Antibody andoligonucleotide biotinylation techniques can be used, and are describedfor example in Fang et al., Nucleic Acids Res. (2003), 31(2): 708-715,the entire contents of which are incorporated by reference herein.Likewise, protein and peptide biotinylation techniques have beendeveloped and can be used, and are described for example in U.S. Pat.No. 6,265,552, the entire contents of which are incorporated byreference herein. Furthermore, click reaction chemistry such as amethyltetrazine-PEG5-NHS ester reaction, a TCO-PEG4-NHS ester reaction,or the like, can be used to couple capture agent barcode domains toanalyte binding moieties. The reactive moiety on the analyte bindingmoiety can also include amine for targeting aldehydes, amine fortargeting maleimide (e.g., free thiols), azide for targeting clickchemistry compounds (e.g., alkynes), biotin for targeting streptavidin,phosphates for targeting EDC, which in turn targets active ester (e.g.,NH2). The reactive moiety on the analyte binding moiety can be achemical compound or group that binds to the reactive moiety on theanalyte binding moiety. Exemplary strategies to conjugate the analytebinding moiety to the capture agent barcode domain include the use ofcommercial kits (e.g., Solulink, Thunder link), conjugation of mildreduction of hinge region and maleimide labelling, stain-promoted clickchemistry reaction to labeled amides (e.g., copper-free), andconjugation of periodate oxidation of sugar chain and amine conjugation.In the cases where the analyte binding moiety is an antibody, theantibody can be modified prior to or contemporaneously with conjugationof the oligonucleotide. For example, the antibody can be glycosylatedwith a chemical substrate-permissive mutant ofβ-1,4-galactosyltransferase, GalT (Y289L) and azide-bearing uridinediphosphate-N-acetylgalactosamine analog uridine diphosphate —GalNAz.The modified antibody can be conjugated to an oligonucleotide with adibenzocyclooctyne-PEG4-NHS group. In some embodiments, certain steps(e.g., COOH activation (e.g., EDC) and homobifunctional cross linkers)can be avoided to prevent the analyte binding moieties from conjugatingto themselves. In some embodiments of any of the spatial profilingmethods described herein, the analyte capture agent (e.g., analytebinding moiety coupled to an oligonucleotide) can be delivered into thecell, e.g., by transfection (e.g., using transfectamine, cationicpolymers, calcium phosphate or electroporation), by transduction (e.g.,using a bacteriophage or recombinant viral vector), by mechanicaldelivery (e.g., magnetic beads), by lipid (e.g.,1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC)), or by transporterproteins. An analyte capture agent can be delivered into a cell usingexosomes. For example, a first cell can be generated that releasesexosomes comprising an analyte capture agent. An analyte capture agentcan be attached to an exosome membrane. An analyte capture agent can becontained within the cytosol of an exosome. Released exosomes can beharvested and provided to a second cell, thereby delivering the analytecapture agent into the second cell. An analyte capture agent can bereleasable from an exosome membrane before, during, or after deliveryinto a cell. In some embodiments, the cell is permeabilized to allow theanalyte capture agent to couple with intracellular constituents (suchas, without limitation, intracellular proteins, metabolites, and nuclearmembrane proteins). Following intracellular delivery, analyte captureagents can be used to analyze intracellular constituents as describedherein.

In some embodiments of any of the spatial profiling methods describedherein, the capture agent barcode domain coupled to an analyte captureagent can include modifications that render it non-extendable by apolymerase. In some embodiments, when binding to a capture domain of acapture probe or nucleic acid in a sample for a primer extensionreaction, the capture agent barcode domain can serve as a template, nota primer. When the capture agent barcode domain also includes a barcode(e.g., an analyte binding moiety barcode), such a design can increasethe efficiency of molecular barcoding by increasing the affinity betweenthe capture agent barcode domain and unbarcoded sample nucleic acids,and eliminate the potential formation of adaptor artifacts. In someembodiments, the capture agent barcode domain can include a random N-mersequence that is capped with modifications that render it non-extendableby a polymerase. In some cases, the composition of the random N-mersequence can be designed to maximize the binding efficiency to free,unbarcoded ssDNA molecules. The design can include a random sequencecomposition with a higher GC content, a partial random sequence withfixed G or C at specific positions, the use of guanosines, the use oflocked nucleic acids, or any combination thereof.

A modification for blocking primer extension by a polymerase can be acarbon spacer group of different lengths or a dideoxynucleotide. In someembodiments, the modification can be an abasic site that has an apurineor apyrimidine structure, a base analog, or an analogue of a phosphatebackbone, such as a backbone of N-(2-aminoethyl)-glycine linked by amidebonds, tetrahydrofuran, or 1′, 2′-Dideoxyribose. The modification canalso be a uracil base, 2′OMe modified RNA, C3-18 spacers (e.g.,structures with 3-18 consecutive carbon atoms, such as C3 spacer),ethylene glycol multimer spacers (e.g., spacer 18 (hexa-ethyleneglycolspacer), biotin, di-deoxynucleotide triphosphate, ethylene glycol,amine, or phosphate.

In some embodiments of any of the spatial profiling methods describedherein, the capture agent barcode domain coupled to the analyte bindingmoiety includes a cleavable domain. For example, after the analytecapture agent binds to an analyte (e.g., a cell surface analyte), thecapture agent barcode domain can be cleaved and collected for downstreamanalysis according to the methods as described herein. In someembodiments, the cleavable domain of the capture agent barcode domainincludes a U-excising element that allows the species to release fromthe bead. In some embodiments, the U-excising element can include asingle-stranded DNA (ssDNA) sequence that contains at least one uracil.The species can be attached to a bead via the ssDNA sequence. Thespecies can be released by a combination of uracil-DNA glycosylase(e.g., to remove the uracil) and an endonuclease (e.g., to induce anssDNA break). If the endonuclease generates a 5′ phosphate group fromthe cleavage, then additional enzyme treatment can be included indownstream processing to eliminate the phosphate group, e.g., prior toligation of additional sequencing handle elements, e.g., Illumina fullP5 sequence, partial P5 sequence, full R1 sequence, and/or partial R1sequence.

In some embodiments, multiple different species of analytes (e.g.,polypeptides) from the biological sample can be subsequently associatedwith the one or more physical properties of the biological sample. Forexample, the multiple different species of analytes can be associatedwith locations of the analytes in the biological sample. Suchinformation (e.g., proteomic information when the analyte bindingmoiety(ies) recognizes a polypeptide(s)) can be used in association withother spatial information (e.g., genetic information from the biologicalsample, such as DNA sequence information, transcriptome information(i.e., sequences of transcripts), or both). For example, a cell surfaceprotein of a cell can be associated with one or more physical propertiesof the cell (e.g., a shape, size, activity, or a type of the cell). Theone or more physical properties can be characterized by imaging thecell. The cell can be bound by an analyte capture agent comprising ananalyte binding moiety that binds to the cell surface protein and ananalyte binding moiety barcode that identifies that analyte bindingmoiety, and the cell can be subjected to spatial analysis (e.g., any ofthe variety of spatial analysis methods described herein). For example,the analyte capture agent bound to the cell surface protein can be boundto a capture probe (e.g., a capture probe on an array), which captureprobe includes a capture domain that interacts with an analyte capturesequence present on the capture agent barcode domain of the analytecapture agent. All or part of the capture agent barcode domain(including the analyte binding moiety barcode) can be copied with apolymerase using a 3′ end of the capture domain as a priming site,generating an extended capture probe that includes the all or part ofcomplementary sequence that corresponds to the capture probe (includinga spatial barcode present on the capture probe) and a copy of theanalyte binding moiety barcode. In some embodiments, an analyte captureagent with an extended capture agent barcode domain that includes asequence complementary to a spatial barcode of a capture probe is calleda “spatially-tagged analyte capture agent.”

In some embodiments, the spatial array with spatially-tagged analytecapture agents can be contacted with a sample, where the analyte captureagent(s) associated with the spatial array capture the targetanalyte(s). The analyte capture agent(s) containing the extended captureprobe(s), which includes a sequence complementary to the spatialbarcode(s) of the capture probe(s) and the analyte binding moietybarcode(s), can then be denatured from the capture probe(s) of thespatial array. This allows the spatial array to be reused. The samplecan be dissociated into non-aggregated cells (e.g., single cells) andanalyzed by the single cell / droplet methods described herein. Thespatially-tagged analyte capture agent can be sequenced to obtain thenucleic acid sequence of the spatial barcode of the capture probe andthe analyte binding moiety barcode of the analyte capture agent. Thenucleic acid sequence of the extended capture probe can thus beassociated with an analyte (e.g., cell surface protein), and in turn,with the one or more physical properties of the cell (e.g., a shape orcell type). In some embodiments, the nucleic acid sequence of theextended capture probe can be associated with an intracellular analyteof a nearby cell, where the intracellular analyte was released using anyof the cell permeabilization or analyte migration techniques describedherein.

In some embodiments of any of the spatial profiling methods describedherein, the capture agent barcode domains released from the analytecapture agents can then be subjected to sequence analysis to identifywhich analyte capture agents were bound to analytes. Based upon thecapture agent barcode domains that are associated with a feature (e.g.,a feature at a particular location) on a spatial array and the presenceof the analyte binding moiety barcode sequence, an analyte profile canbe created for a biological sample. Profiles of individual cells orpopulations of cells can be compared to profiles from other cells, e.g.,‘normal’ cells, to identify variations in analytes, which can providediagnostically relevant information. In some embodiments, these profilescan be useful in the diagnosis of a variety of disorders that arecharacterized by variations in cell surface receptors, such as cancerand other disorders.

FIG. 10 is a schematic diagram depicting an exemplary interactionbetween a feature-immobilized capture probe 1024 and an analyte captureagent 1026. The feature-immobilized capture probe 1024 can include aspatial barcode 1008 as well as one or more functional sequences 1006and 1010, as described elsewhere herein. The capture probe can alsoinclude a capture domain 1012 that is capable of binding to an analytecapture agent 1026. The analyte capture agent 1026 can include afunctional sequence 1018, capture agent barcode domain 1016, and ananalyte capture sequence 1014 that is capable of binding to the capturedomain 1012 of the capture probe 1024. The analyte capture agent canalso include a linker 1020 that allows the capture agent barcode domain1016 to couple to the analyte binding moiety 1022.

In some embodiments of any of the spatial profiling methods describedherein, the methods are used to identify immune cell profiles. Immunecells express various adaptive immunological receptors relating toimmune function, such as T cell receptors (TCRs) and B cell receptors(BCRs). T cell receptors and B cell receptors play a part in the immuneresponse by specifically recognizing and binding to antigens and aidingin their destruction.

The T cell receptor, or TCR, is a molecule found on the surface of Tcells that is generally responsible for recognizing fragments of antigenas peptides bound to major histocompatibility complex (MHC) molecules.The TCR is generally a heterodimer of two chains, each of which is amember of the immunoglobulin superfamily, possessing an N-terminalvariable (V) domain, and a C terminal constant domain. In humans, in 95%of T cells, the TCR consists of an alpha (α) and beta (β) chain, whereasin 5% of T cells, the TCR consists of gamma and delta (γ/δ) chains. Thisratio can change during ontogeny and in diseased states as well as indifferent species. When the TCR engages with antigenic peptide and MHC(peptide/MHC or pMHC), the T lymphocyte is activated through signaltransduction.

Each of the two chains of a TCR contains multiple copies of genesegments - a variable ‘V’ gene segment, a diversity ‘D’ gene segment,and a joining ‘J’ gene segment. The TCR alpha chain (TCRa) is generatedby recombination of V and J segments, while the beta chain (TCRb) isgenerated by recombination of V, D, and J segments. Similarly,generation of the TCR gamma chain involves recombination of V and J genesegments, while generation of the TCR delta chain occurs byrecombination of V, D, and J gene segments. The intersection of thesespecific regions (V and J for the alpha or gamma chain, or V, D and Jfor the beta or delta chain) corresponds to the CDR3 region that isimportant for antigen-MHC recognition. Complementarity determiningregions (e.g., CDR1, CDR2, and CDR3), or hypervariable regions, aresequences in the variable domains of antigen receptors (e.g., T cellreceptor and immunoglobulin) that can complement an antigen. Most of thediversity of CDRs is found in CDR3, with the diversity being generatedby somatic recombination events during the development of T lymphocytes.A unique nucleotide sequence that arises during the gene arrangementprocess can be referred to as a clonotype.

The B cell receptor, or BCR, is a molecule found on the surface of Bcells. The antigen binding portion of a BCR is composed of amembrane-bound antibody that, like most antibodies (e.g.,immunoglobulins), has a unique and randomly determined antigen-bindingsite. The antigen binding portion of a BCR includes membrane-boundimmunoglobulin molecule of one isotype (e.g., IgD, IgM, IgA, IgG, orIgE). When a B cell is activated by its first encounter with a cognateantigen, the cell proliferates and differentiates to generate apopulation of antibody-secreting plasma B cells and memory B cells. Thevarious immunoglobulin isotypes differ in their biological features,structure, target specificity, and distribution. A variety of molecularmechanisms exist to generate initial diversity, including geneticrecombination at multiple sites.

The BCR is composed of two genes IgH and IgK (or IgL) coding forantibody heavy and light chains. Immunoglobulins are formed byrecombination among gene segments, sequence diversification at thejunctions of these segments, and point mutations throughout the gene.Each heavy chain gene contains multiple copies of three different genesegments - a variable ‘V’ gene segment, a diversity ‘D’ gene segment,and a joining ‘J’ gene segment. Each light chain gene contains multiplecopies of two different gene segments for the variable region of theprotein - a variable ‘V’ gene segment and a joining ‘J’ gene segment.

The recombination can generate a molecule with one of each of the V, D,and J segments. Furthermore, several bases can be deleted and othersadded (called N and P nucleotides) at each of the two junctions, therebygenerating further diversity. After B cell activation, a process ofaffinity maturation through somatic hypermutation occurs. In thisprocess, progeny cells of the activated B cells accumulate distinctsomatic mutations throughout the gene with higher mutation concentrationin the CDR regions leading to the generation of antibodies with higheraffinity to the antigens.

In addition to somatic hypermutation, activated B cells undergo theprocess of isotype switching. Antibodies with the same variable segmentscan have different forms (isotypes) depending on the constant segment.Whereas all naïve B cells express IgM (or IgD), activated B cells mostlyexpress IgG but also IgM, IgA, and IgE. This expression switching fromIgM (and/or IgD) to IgG, IgA, or IgE occurs through a recombinationevent causing one cell to specialize in producing a specific isotype. Aunique nucleotide sequence that arises during the gene arrangementprocess can similarly be referred to as a clonotype.

Certain methods described herein are utilized to analyze the varioussequences of TCRs and BCRs from immune cells, for example, variousclonotypes. In some embodiments, the methods are used to analyze thesequence of a TCR alpha chain, a TCR beta chain, a TCR delta chain, aTCR gamma chain, or any fragment thereof (e.g., variable regionsincluding V(D)J or VJ regions, constant regions, transmembrane regions,fragments thereof, combinations thereof, and combinations of fragmentsthereof). In some embodiments, the methods described herein can be usedto analyze the sequence of a B cell receptor heavy chain, B cellreceptor light chain, or any fragment thereof (e.g., variable regionsincluding V(D)J or VJ regions, constant regions, transmembrane regions,fragments thereof, combinations thereof, and combinations of fragmentsthereof).

Where immune cells are to be analyzed, primer sequences useful in any ofthe various operations for attaching barcode sequences and/oramplification reactions can include gene specific sequences which targetgenes or regions of genes of immune cell proteins, for example immunereceptors. Such gene sequences include, but are not limited to,sequences of various T cell receptor alpha variable genes (TRAV genes),T cell receptor alpha joining genes (TRAJ genes), T cell receptor alphaconstant genes (TRAC genes), T cell receptor beta variable genes (TRBVgenes), T cell receptor beta diversity genes (TRBD genes), T cellreceptor beta joining genes (TRBJ genes), T cell receptor beta constantgenes (TRBC genes), T cell receptor gamma variable genes (TRGV genes), Tcell receptor gamma joining genes (TRGJ genes), T cell receptor gammaconstant genes (TRGC genes), T cell receptor delta variable genes (TRDVgenes), T cell receptor delta diversity genes (TRDD genes), T cellreceptor delta joining genes (TRDJ genes), and T cell receptor deltaconstant genes (TRDC genes).

In some embodiments, the analyte binding moiety is based on the MajorHistocompatibility Complex (MHC) class I or class II. In someembodiments, the analyte binding moiety is an MHC multimer including,without limitation, MHC dextramers, MHC tetramers, and MHC pentamers(see, for example, U.S. Pat. Application Publication Nos. US2018/0180601 and US 2017/0343545, the entire contents of each of whichare incorporated herein by reference. MHCs (e.g., a soluble MHC monomermolecule), including full or partial MHC-peptides, can be used asanalyte binding moieties of analyte capture agents that are coupled tocapture agent barcode domains that include an analyte binding moietybarcode that identifies its associated MHC (and, thus, for example, theMHC’s TCR binding partner). In some embodiments, MHCs are used toanalyze one or more cell-surface features of a T-cell, such as a TCR. Insome cases, multiple MHCs are associated together in a larger complex(MHC multi-mer) to improve binding affinity of MHCs to TCRs via multipleligand binding synergies.

FIGS. 11A, 11B, and 11C are schematics illustrating how streptavidincell tags can be utilized in an array-based system to produce aspatially-barcoded cell or cellular contents. For example, as shown inFIG. 11 , peptide-bound major histocompatibility complex (pMHCs) can beindividually associated with biotin and bound to a streptavidin moietysuch that the streptavidin moiety comprises multiple pMHC moieties. Eachof these moieties can bind to a TCR such that the streptavidin binds toa target T-cell via multiple MCH/TCR binding interactions. Multipleinteractions synergize and can substantially improve binding affinity.Such improved affinity can improve labelling of T-cells and also reducethe likelihood that labels will dissociate from T-cell surfaces. Asshown in FIG. 11B, a capture agent barcode domain 1101 can be modifiedwith streptavidin 1102 and contacted with multiple molecules ofbiotinylated MHC 1103 (such as a pMHC) such that the biotinylated MHC1103 molecules are coupled with the streptavidin conjugated captureagent barcode domain 1101. The result is a barcoded MHC multimer complex1105. As shown in FIG. 11B, the capture agent barcode domain sequence1101 can identify the MHC as its associated label and also includesoptional functional sequences such as sequences for hybridization withother oligonucleotides. As shown in FIG. 11C, one exampleoligonucleotide is capture probe 1106 that comprises a complementarysequence (e.g., rGrGrG corresponding to C C C), a barcode sequence andother functional sequences, such as, for example, a UMI, an adaptersequence (e.g., comprising a sequencing primer sequence (e.g., R1 or apartial R1 (“pR1”)), a flow cell attachment sequence (e.g., P5 or P7 orpartial sequences thereof)), etc. In some cases, capture probe 1106 mayat first be associated with a feature (e.g., a gel bead) and releasedfrom the feature. In other embodiments, capture probe 1106 can hybridizewith a capture agent barcode domain 1101 of the MHC-oligonucleotidecomplex 1105. The hybridized oligonucleotides (Spacer C C C and SpacerrGrGrG) can then be extended in primer extension reactions such thatconstructs comprising sequences that correspond to each of the twospatial barcode sequences (the spatial barcode associated with thecapture probe, and the barcode associated with the MHC-oligonucleotidecomplex) are generated. In some cases, one or both of thesecorresponding sequences may be a complement of the original sequence incapture probe 1106 or capture agent barcode domain 1101. In otherembodiments, the capture probe and the capture agent barcode domain areligated together. The resulting constructs can be optionally furtherprocessed (e.g., to add any additional sequences and/or for clean-up)and subjected to sequencing. As described elsewhere herein, a sequencederived from the capture probe 1106 spatial barcode sequence may be usedto identify a feature and the sequence derived from spatial barcodesequence on the capture agent barcode domain 1101 may be used toidentify the particular peptide MHC complex 1104 bound on the surface ofthe cell (e.g., when using MHC-peptide libraries for screening immunecells or immune cell populations).

(C) Substrates

For the spatial array-based analytical methods described herein, asubstrate functions as a support for direct or indirect attachment ofcapture probes to features of the array. In addition, in someembodiments, a substrate (e.g., the same substrate or a differentsubstrate) can be used to provide support to a biological sample,particularly, for example, a thin tissue section. Accordingly, a“substrate” is a support that is insoluble in aqueous liquid and whichallows for positioning of biological samples, analytes, features, and/orcapture probes on the substrate.

Further, a “substrate” as used herein, and when not preceded by themodifier “chemical”, refers to a member with at least one surface thatgenerally functions to provide physical support for biological samples,analytes, and/or any of the other chemical and/or physical moieties,agents, and structures described herein. Substrates can be formed from avariety of solid materials, gel-based materials, colloidal materials,semi-solid materials (e.g., materials that are at least partiallycross-linked), materials that are fully or partially cured, andmaterials that undergo a phase change or transition to provide physicalsupport. Examples of substrates that can be used in the methods andsystems described herein include, but are not limited to, slides (e.g.,slides formed from various glasses, slides formed from variouspolymers), hydrogels, layers and/or films, membranes (e.g., porousmembranes), flow cells, cuvettes, wafers, plates, or combinationsthereof. In some embodiments, substrates can optionally includefunctional elements such as recesses, protruding structures,microfluidic elements (e.g., channels, reservoirs, electrodes, valves,seals), and various markings, as will be discussed in further detailbelow.

(I) Substrate Attributes

A substrate can generally have any suitable form or format. For example,a substrate can be flat, curved, e.g., convexly or concavely curvedtowards the area where the interaction between a biological sample,e.g., tissue sample, and a substrate takes place. In some embodiments, asubstrate is flat, e.g., planar, chip, or slide. A substrate can containone or more patterned surfaces within the substrate (e.g., channels,wells, projections, ridges, divots, etc.).

A substrate can be of any desired shape. For example, a substrate can betypically a thin, flat shape (e.g., a square or a rectangle). In someembodiments, a substrate structure has rounded corners (e.g., forincreased safety or robustness). In some embodiments, a substratestructure has one or more cut-off corners (e.g., for use with a slideclamp or cross-table). In some embodiments, where a substrate structureis flat, the substrate structure can be any appropriate type of supporthaving a flat surface (e.g., a chip or a slide such as a microscopeslide).

Substrates can optionally include various structures such as, but notlimited to, projections, ridges, and channels. A substrate can bemicropatterned to limit lateral diffusion (e.g., to prevent overlap ofspatial barcodes). A substrate modified with such structures can bemodified to allow association of analytes, features (e.g., beads), orprobes at individual sites. For example, the sites where a substrate ismodified with various structures can be contiguous or non-contiguouswith other sites.

In some embodiments, the surface of a substrate can be modified so thatdiscrete sites are formed that can only have or accommodate a singlefeature. In some embodiments, the surface of a substrate can be modifiedso that features adhere to random sites.

In some embodiments, the surface of a substrate is modified to containone or more wells, using techniques such as (but not limited to)stamping, microetching, or molding techniques. In some embodiments inwhich a substrate includes one or more wells, the substrate can be aconcavity slide or cavity slide. For example, wells can be formed by oneor more shallow depressions on the surface of the substrate. In someembodiments, where a substrate includes one or more wells, the wells canbe formed by attaching a cassette (e.g., a cassette containing one ormore chambers) to a surface of the substrate structure.

In some embodiments, the structures of a substrate (e.g., wells orfeatures) can each bear a different capture probe. Different captureprobes attached to each structure can be identified according to thelocations of the structures in or on the surface of the substrate.Exemplary substrates include arrays in which separate structures arelocated on the substrate including, for example, those having wells thataccommodate features.

In some embodiments where the substrate is modified to contain one ormore structures, including but not limited to, wells, projections,ridges, features, or markings, the structures can include physicallyaltered sites. For example, a substrate modified with various structurescan include physical properties, including, but not limited to, physicalconfigurations, magnetic or compressive forces, chemicallyfunctionalized sites, chemically altered sites, and/or electrostaticallyaltered sites. In some embodiments where the substrate is modified tocontain various structures, including but not limited to wells,projections, ridges, features, or markings, the structures are appliedin a pattern. Alternatively, the structures can be randomly distributed.

The substrate (e.g., or a bead or a feature on an array) can includetens to hundreds of thousands or millions of individual oligonucleotidemolecules (e.g., at least about 10,000, 50,000, 100,000, 500,000,1,000,000, 10,000,000, 100,000,000, 1,000,000,000, or 10,000,000,000oligonucleotide molecules).

In some embodiments, a substrate includes one or more markings on asurface of a substrate, e.g., to provide guidance for correlatingspatial information with the characterization of the analyte ofinterest. For example, a substrate can be marked with a grid of lines(e.g., to allow the size of objects seen under magnification to beeasily estimated and/or to provide reference areas for countingobjects). In some embodiments, fiducial markers can be included on asubstrate. Such markings can be made using techniques including, but notlimited to, printing, sand-blasting, and depositing on the surface.

In some embodiments, imaging can be performed using one or more fiducialmarkers, i.e., objects placed in the field of view of an imaging systemwhich appear in the image produced. Fiducial markers are typically usedas a point of reference or measurement scale. Fiducial markers caninclude, but are not limited to, detectable labels such as fluorescent,radioactive, chemiluminescent, and colorimetric labels. The use offiducial markers to stabilize and orient biological samples isdescribed, for example, in Carter et al., Applied Optics 46:421-427,2007), the entire contents of which are incorporated herein byreference. In some embodiments, a fiducial marker can be a physicalparticle (e.g., a nanoparticle, a microsphere, a nanosphere, a bead, orany of the other exemplary physical particles described herein or knownin the art).

In some embodiments, a fiducial marker can be present on a substrate toprovide orientation of the biological sample. In some embodiments, amicrosphere can be coupled to a substrate to aid in orientation of thebiological sample. In some examples, a microsphere coupled to asubstrate can produce an optical signal (e.g., fluorescence). In anotherexample, a microsphere can be attached to a portion (e.g., corner) of anarray in a specific pattern or design (e.g., hexagonal design) to aid inorientation of a biological sample on an array of features on thesubstrate. In some embodiments, a quantum dot can be coupled to thesubstrate to aid in the orientation of the biological sample. In someexamples, a quantum dot coupled to a substrate can produce an opticalsignal.

In some embodiments, a fiducial marker can be an immobilized moleculewith which a detectable signal molecule can interact to generate asignal. For example, a marker nucleic acid can be linked or coupled to achemical moiety capable of fluorescing when subjected to light of aspecific wavelength (or range of wavelengths). Such a marker nucleicacid molecule can be contacted with an array before, contemporaneouslywith, or after the tissue sample is stained to visualize or image thetissue section. Although not required, it can be advantageous to use amarker that can be detected using the same conditions (e.g., imagingconditions) used to detect a labelled cDNA.

In some embodiments, fiducial markers are included to facilitate theorientation of a tissue sample or an image thereof in relation to animmobilized capture probes on a substrate. Any number of methods formarking an array can be used such that a marker is detectable only whena tissue section is imaged. For instance, a molecule, e.g., afluorescent molecule that generates a signal, can be immobilizeddirectly or indirectly on the surface of a substrate. Markers can beprovided on a substrate in a pattern (e.g., an edge, one or more rows,one or more lines, etc.).

In some embodiments, a fiducial marker can be randomly placed in thefield of view. For example, an oligonucleotide containing a fluorophorecan be randomly printed, stamped, synthesized, or attached to asubstrate (e.g., a glass slide) at a random position on the substrate. Atissue section can be contacted with the substrate such that theoligonucleotide containing the fluorophore contacts, or is in proximityto, a cell from the tissue section or a component of the cell (e.g., anmRNA or DNA molecule). An image of the substrate and the tissue sectioncan be obtained, and the position of the fluorophore within the tissuesection image can be determined (e.g., by reviewing an optical image ofthe tissue section overlaid with the fluorophore detection). In someembodiments, fiducial markers can be precisely placed in the field ofview (e.g., at known locations on a substrate). In this instance, afiducial marker can be stamped, attached, or synthesized on thesubstrate and contacted with a biological sample. Typically, an image ofthe sample and the fiducial marker is taken, and the position of thefiducial marker on the substrate can be confirmed by viewing the image.

In some embodiments, a fiducial marker can be an immobilized molecule(e.g., a physical particle) attached to the substrate. For example, afiducial marker can be a nanoparticle, e.g., a nanorod, a nanowire, ananocube, a nanopyramid, or a spherical nanoparticle. In some examples,the nanoparticle can be made of a heavy metal (e.g., gold). In someembodiments, the nanoparticle can be made from diamond. In someembodiments, the fiducial marker can be visible by eye.

As noted herein, any of the fiducial markers described herein (e.g.,microspheres, beads, or any of the other physical particles describedherein) can be located at a portion (e.g., corner) of an array in aspecific pattern or design (e.g., hexagonal design) to aid inorientation of a biological sample on an array of features on thesubstrate. In some embodiments, the fiducial markers located at aportion (e.g., corner) of an array (e.g., an array on a substrate) canbe patterned or designed in at least 1, at least 2, at least 3, or atleast 4 unique patterns. In some examples, the fiducial markers locatedat the corners of the array (e.g., an array on a substrate) can havefour unique patterns of fiducial markers.

In some examples, fiducial markers can surround the array. In someembodiments the fiducial markers allow for detection of, e.g.,mirroring. In some embodiments, the fiducial markers may completelysurround the array. In some embodiments, the fiducial markers may notcompletely surround the array. In some embodiments, the fiducial markersidentify the corners of the array. In some embodiments, one or morefiducial markers identify the center of the array. In some embodiments,the fiducial markers comprise patterned spots, wherein the diameter ofone or more patterned spot fiducial markers is approximately 100micrometers. The diameter of the fiducial markers can be any usefuldiameter including, but not limited to, 50 micrometers to 500micrometers in diameter. The fiducial markers may be arranged in such away that the center of one fiducial marker is between 100 micrometersand 200 micrometers from the center of one or more other fiducialmarkers surrounding the array. In some embodiments, the array with thesurrounding fiducial markers is approximately 8 mm by 8 mm. In someembodiments, the array without the surrounding fiducial markers issmaller than 8 mm by 50 mm.

In some embodiments, an array can be enclosed within a frame. Putanother way, the perimeter of an array can have fiducial markers suchthat the array is enclosed, or substantially enclosed. In someembodiments, the perimeter of an array can be fiducial markers (e.g.,any fiducial marker described herein). In some embodiments, theperimeter of an array can be uniform. For example, the fiducial markingscan connect, or substantially connect, consecutive corners of an arrayin such a fashion that the non-corner portion of the array perimeter isthe same on all sides (e.g., four sides) of the array. In someembodiments, the fiducial markers attached to the non-corner portions ofthe perimeter can be pattered or designed to aid in the orientation ofthe biological sample on the array. In some embodiments, the particlesattached to the non-corner portions of the perimeter can be patterned ordesigned in at least 1, at least 2, at least 3, or at least 4 patterns.In some embodiments, the patterns can have at least 2, at least 3, or atleast 4 unique patterns of fiducial markings on the non-corner portionof the array perimeter.

In some embodiments, an array can include at least two fiducial markers(e.g., at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 12, at least 15, at least 20,at least 30, at least 40, at least 50, at least 60, at least 70, atleast 80, at least 90, at least 100 fiducial markers or more (e.g.,several hundred, several thousand, or tens of thousands of fiducialmarkers)) in distinct positions on the surface of a substrate. Fiducialmarkers can be provided on a substrate in a pattern (e.g., an edge, oneor more rows, one or more lines, etc.).

A wide variety of different substrates can be used for the foregoingpurposes. In general, a substrate can be any suitable support material.Exemplary substrates include, but are not limited to, glass, modifiedand/or functionalized glass, hydrogels, films, membranes, plastics(including e.g., acrylics, polystyrene, copolymers of styrene and othermaterials, polypropylene, polyethylene, polybutylene, polyurethanes,Teflon™, cyclic olefins, polyimides etc.), nylon, ceramics, resins,Zeonor, silica or silica-based materials including silicon and modifiedsilicon, carbon, metals, inorganic glasses, optical fiber bundles, andpolymers, such as polystyrene, cyclic olefin copolymers (COCs), cyclicolefin polymers (COPs), polypropylene, polyethylene polycarbonate, orcombinations thereof.

Among the examples of substrate materials discussed above, polystyreneis a hydrophobic material suitable for binding negatively chargedmacromolecules because it normally contains few hydrophilic groups. Fornucleic acids immobilized on glass slides, by increasing thehydrophobicity of the glass surface the nucleic acid immobilization canbe increased. Such an enhancement can permit a relatively more denselypacked formation (e.g., provide improved specificity and resolution).

In another example, a substrate can be a flow cell. Flow cells can beformed of any of the foregoing materials, and can include channels thatpermit reagents, solvents, features, and analytes to pass through theflow cell. In some embodiments, a hydrogel embedded biological sample isassembled in a flow cell (e.g., the flow cell is utilized to introducethe hydrogel to the biological sample). In some embodiments, a hydrogelembedded biological sample is not assembled in a flow cell. In someembodiments, the hydrogel embedded biological sample can then beprepared and/or isometrically expanded as described herein.

(II) Conductive Substrates

Conductive substrates (e.g., electrophoretic compatible arrays)generated as described herein can be used in the spatial detection ofanalytes. For example, an electrophoretic field can be applied tofacilitate migration of analytes towards the barcoded oligonucleotides(e.g., capture probes) on the array (e.g., capture probes immobilized onpaper, capture probes immobilized in a hydrogel film, or capture probesimmobilized on a glass slide having a conductive coating). In someembodiments, an electrophoresis assembly can be arranged. For example,an anode and a cathode can be arranged such that an array of captureprobes (e.g., capture probes immobilized on paper, capture probesimmobilized in a hydrogel film, or capture probes immobilized on a glassslide having a conductive coating) and a biological sample arepositioned between the anode and the cathode. In such embodiments,analytes in the biological sample are actively migrated toward thecapture probes on the conductive substrate and captured. The biologicalsample can be prepared (e.g., permeabilized) according to any methoddescribed herein. In some embodiments, after electrophoretic-assistedcapture of the analytes, the barcoded oligonucleotides (e.g., captureprobes) and captured analytes can be collected, processed, and/oranalyzed (e.g., sequenced) using any of the methods described herein.

In some embodiments, a conductive substrate can include glass (e.g., aglass slide) that has been coated with a substance or otherwise modifiedto confer conductive properties to the glass. In some embodiments, aglass slide can be coated with a conductive coating. In someembodiments, a conductive coating includes tin oxide (TO) or indium tinoxide (ITO). In some embodiments, a conductive coating includes atransparent conductive oxide (TCO). In some embodiments, a conductivecoating includes aluminum doped zinc oxide (AZO). In some embodiments, aconductive coating includes fluorine doped tin oxide (FTO).

In some embodiments, arrays that are spotted or printed witholigonucleotides (e.g., capture probes, e.g., any of the variety ofcapture probes described herein) can be generated on a conductivesubstrate (e.g., any of the conductive substrates described herein). Forexample, the arrays described herein can be compatible with activeanalyte capture methods (e.g., any of the analyte capture methodsdescribed herein, including without limitation, electrophoretic capturemethods). In some embodiments, a conductive substrate is a porousmedium. Non-limiting examples of porous media that can be used inmethods described herein that employ active analyte capture include anitrocellulose or nylon membrane. In some embodiments, a porous mediumthat can be used in methods described herein that employ active analytecapture includes paper. In some embodiments, the oligonucleotides can beprinted on a paper substrate. In some embodiments, the printedoligonucleotides can interact with the substrate (e.g., interact withfibers of the paper). In some embodiments, printed oligonucleotides cancovalently bind the substrate (e.g., to fibers of the paper). In someembodiments, oligonucleotides in a molecular precursor solution can beprinted on a conductive substrate (e.g., paper). In some embodiments, amolecular precursor solution can polymerize, thereby generating gel padson the conductive substrate (e.g., paper). In some embodiments, amolecular precursor solution can be polymerized by light (e.g.,photocured). In some embodiments, gel beads (e.g., any of the variety ofgel beads described herein) containing oligonucleotides (e.g., barcodedoligonucleotides such as capture probes) can be printed on a conductivesubstrate (e.g., paper). In some embodiments, the printedoligonucleotides can be covalently attached into the gel matrix.

(III) Coatings

In some embodiments, a surface of a substrate can be coated with acell-permissive coating to allow adherence of live cells. A“cell-permissive coating” is a coating that allows or helps cells tomaintain cell viability (e.g., remain viable) on the substrate. Forexample, a cell-permissive coating can enhance cell attachment, cellgrowth, and/or cell differentiation, e.g., a cell-permissive coating canprovide nutrients to the live cells. A cell-permissive coating caninclude a biological material and/or a synthetic material. Non-limitingexamples of a cell-permissive coating include coatings that feature oneor more extracellular matrix (ECM) components (e.g., proteoglycans andfibrous proteins such as collagen, elastin, fibronectin and laminin),poly-lysine, poly(L)-ornithine, and/or a biocompatible silicone (e.g.,CYTOSOFT®). For example, a cell-permissive coating that includes one ormore extracellular matrix components can include collagen Type I,collagen Type II, collagen Type IV, elastin, fibronectin, laminin,and/or vitronectin. In some embodiments, the cell-permissive coatingincludes a solubilized basement membrane preparation extracted from theEngelbreth-Holm-Swarm (EHS) mouse sarcoma (e.g., MATRIGEL®). In someembodiments, the cell-permissive coating includes collagen. Acell-permissive coating can be used to culture adherent cells on aspatially-barcoded array, or to maintain cell viability of a tissuesample or section while in contact with a spatially-barcoded array.

In some embodiments, a substrate is coated with a surface treatment suchas poly(L)-lysine. Additionally or alternatively, the substrate can betreated by silanation, e.g., with epoxy-silane, amino-silane, and/or bya treatment with polyacrylamide.

In some embodiments, a substrate is treated in order to minimize orreduce non-specific analyte hybridization within or between features.For example, treatment can include coating the substrate with ahydrogel, film, and/or membrane that creates a physical barrier tonon-specific hybridization. Any suitable hydrogel can be used. Forexample, hydrogel matrices prepared according to the methods set forthin U.S. Pat. Nos. 6,391,937, 9,512,422, and 9,889,422, and U.S. Pat.Application Publication Nos. U.S. 2017/0253918 and U.S. 2018/0052081,can be used. The entire contents of each of the foregoing documents isincorporated herein by reference.

Treatment can include adding a functional group that is reactive orcapable of being activated such that it becomes reactive afterapplication of a stimulus (e.g., photoreactive functional groups).Treatment can include treating with polymers having one or more physicalproperties (e.g., mechanical, electrical, magnetic, and/or thermal) thatminimize non-specific binding (e.g., that activate a substrate atcertain locations to allow analyte hybridization at those locations).

A “conditionally removable coating” is a coating that can be removedfrom the surface of a substrate upon application of a releasing agent.In some embodiments, a conditionally removable coating includes ahydrogel as described herein, e.g., a hydrogel including apolypeptide-based material. Non-limiting examples of a hydrogelfeaturing a polypeptide-based material include a synthetic peptide-basedmaterial featuring a combination of spider silk and a trans-membranesegment of human muscle L-type calcium channel (e.g., PEPGEL®), anamphiphilic 16 residue peptide containing a repeatingarginine-alanine-aspartate-alanine sequence (RADARADARADARADA) (e.g.,PURAMATRIX®), EAK16 (AEAEAKAKAEAEAKAK), KLD12 (KLDLKLDLKLDL), andPGMATRIX™.

In some embodiments, the hydrogel in the conditionally removable coatingis a stimulus-responsive hydrogel. A stimulus-responsive hydrogel canundergo a gel-to-solution and/or gel-to-solid transition uponapplication of one or more external triggers (e.g., a releasing agent).See, e.g., Willner, Acc. Chem. Res. 50:657-658, 2017, which isincorporated herein by reference in its entirety. Non-limiting examplesof a stimulus-responsive hydrogel include a thermoresponsive hydrogel, apH-responsive hydrogel, a light-responsive hydrogel, a redox-responsivehydrogel, an analyte-responsive hydrogel, or a combination thereof. Insome embodiments, a stimulus-responsive hydrogel can be amulti-stimuli-responsive hydrogel.

A “releasing agent” or “external trigger” is an agent that allows forthe removal of a conditionally removable coating from a substrate whenthe releasing agent is applied to the conditionally removable coating.An external trigger or releasing agent can include physical triggerssuch as thermal, magnetic, ultrasonic, electrochemical, and/or lightstimuli as well as chemical triggers such as pH, redox reactions,supramolecular complexes, and/or biocatalytically driven reactions. Seee.g., Echeverria, et al., Gels (2018), 4, 54; doi:10.3390/gels4020054,which is incorporated herein by reference in its entirety. The type of“releasing agent” or “external trigger” can depend on the type ofconditionally removable coating. For example, a conditionally removablecoating featuring a redox-responsive hydrogel can be removed uponapplication of a releasing agent that includes a reducing agent such asdithiothreitol (DTT). As another example, a pH-responsive hydrogel canbe removed upon the application of a releasing agent that changes thepH.

(IV) Gel Substrates

In some embodiments, a hydrogel can form a substrate. The term“hydrogel” herein refers to a macromolecular polymer gel including anetwork. Within the network, some polymer chains can optionally becross-linked, although cross-linking does not always occur. In someembodiments, the substrate includes a hydrogel and one or more secondmaterials. In some embodiments, the hydrogel is placed on top of one ormore second materials. For example, the hydrogel can be pre-formed andthen placed on top of, underneath, or in any other configuration withone or more second materials. In some embodiments, hydrogel formationoccurs after contacting one or more second materials during formation ofthe substrate. Hydrogel formation can also occur within a structure(e.g., wells, ridges, features, projections, and/or markings) located ona substrate. Where the substrate includes a gel (e.g., a hydrogel or gelmatrix), oligonucleotides within the gel can attach to the substrate.

In some embodiments, a hydrogel can include hydrogel subunits. Thehydrogel subunits can include any convenient hydrogel subunits, such as,but not limited to, acrylamide, bis-acrylamide, polyacrylamide andderivatives thereof, poly(ethylene glycol) and derivatives thereof(e.g., PEG-acrylate (PEG-DA), PEG-RGD), gelatin-methacryloyl (GelMA),methacrylated hyaluronic acid (MeHA), polyaliphatic polyurethanes,polyether polyurethanes, polyester polyurethanes, polyethylenecopolymers, polyamides, polyvinyl alcohols, polypropylene glycol,polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide,poly(hydroxyethyl acrylate), and poly(hydroxyethyl methacrylate),collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin,alginate, protein polymers, methylcellulose, and the like, orcombinations thereof.

In some embodiments, a hydrogel includes a hybrid material, e.g., thehydrogel material includes elements of both synthetic and naturalpolymers. Examples of suitable hydrogels are described, for example, inU.S. Pat. Nos. 6,391,937, 9,512,422, and 9,889,422, and in U.S. Pat.Application Publication Nos. 2017/0253918, 2018/0052081 and2010/0055733, the entire contents of each of which is incorporatedherein by reference.

In some embodiments, cross-linkers and/or initiators are added tohydrogel subunits. Examples of cross-linkers include, withoutlimitation, bis-acrylamide and diazirine. Examples of initiatorsinclude, without limitation, azobisisobutyronitrile (AIBN), riboflavin,and L-arginine. Inclusion of cross-linkers and/or initiators can lead toincreased covalent bonding between interacting biological macromoleculesin later polymerization steps.

In some embodiments, hydrogels can have a colloidal structure, such asagarose, or a polymer mesh structure, such as gelatin. In someembodiments, the hydrogel is a homopolymeric hydrogel. In someembodiments, the hydrogel is a copolymeric hydrogel. In someembodiments, the hydrogel is a multipolymer interpenetrating polymerichydrogel.

In some embodiments, some hydrogel subunits are polymerized (e.g.,undergo “formation”) covalently or physically cross-linked, to form ahydrogel network. For example, hydrogel subunits can be polymerized byany method including, but not limited to, thermal crosslinking, chemicalcrosslinking, physical crosslinking, ionic crosslinking,photocrosslinking, free-radical initiation crosslinking, an additionreaction, condensation reaction, water-soluble crosslinking reactions,irradiative crosslinking (e.g., x-ray, electron beam), or combinationsthereof. Techniques such as lithographic photopolymerization can also beused to form hydrogels.

In some embodiments, gel beads containing oligonucleotides (e.g.,barcoded oligonucleotides such as capture probes) can be deposited on asubstrate (e.g., a glass slide). In some embodiments, gel pads can bedeposited on a substrate (e.g., a glass slide). In some embodiments, gelpads or gel beads are deposited on a substrate in an arrayed format. Insome embodiments in which gel pads or gel beads are deposited on asubstrate in an arrayed format, a hydrogel molecular precursor solutioncan be applied on top of the array (e.g., the array of gel pads or gelbeads on a glass slide). In some embodiments, a hydrogel molecularprecursor solution can be polymerized such that the deposited gel padsor gel beads are immobilized within the polymerized hydrogel. Anysuitable method of polymerization can be used or (e.g., any of thevariety of methods described herein). In some embodiments, a polymerizedhydrogel that includes the gel pads or gel beads can be removed (e.g.,peeled) from the substrate (e.g., glass slide) such that the gel beadsor gel pads are secured in the hydrogel. In some embodiments, apolymerized hydrogel that includes the gel pads or gel beads is aconductive substrate (as described herein) that can be used inaccordance with any of the variety of analyte capture methods describedherein (e.g., electrophoretic migration of analytes for capture).

Arrays can be prepared by depositing features (e.g., droplets, beads) ona substrate surface to produce a spatially-barcoded array. Methods ofdepositing (e.g., droplet manipulation) features are known in the art(see, U.S. Pat. Application Publication No. 2008/0132429, Rubina, A.Y.,et al., Biotechniques. 2003 May; 34(5):1008-14, 1016-20, 1022 andVasiliskov et al. Biotechniques. 1999 September; 27(3):592-4, 596-8, 600passim. each herein incorporated by reference in its entirety). Afeature can be printed or deposited at a specific location on thesubstrate (e.g., inkjet printing). In some embodiments, each feature canhave a unique oligonucleotide that functions as a spatial barcode. Insome embodiments, each feature can have capture probes for multiplexing(e.g., capturing multiple analytes or multiple types of analytes, e.g.,proteins and nucleic acids). In some embodiments, a feature can beprinted or deposited at the specific location using an electric field. Afeature can contain a photo-crosslinkable polymer precursor and anoligonucleotide. In some embodiments, the photo-crosslinkable polymerprecursor can be deposited into a patterned feature on the substrate(e.g., well).

A “photo-crosslinkable polymer precursor” refers to a compound thatcross-links and/or polymerizes upon exposure to light. In someembodiments, one or more photoinitiators may also be included to induceand/or promote polymerization and/or cross-linking. See, e.g., Choi etal. Biotechniques. 2019 Jan;66(1):40-53, which is incorporated herein byreference in its entirety.

Non-limiting examples of photo-crosslinkable polymer precursors includepolyethylene (glycol) diacrylate (PEGDA), gelatin-methacryloyl (GelMA),and methacrylated hyaluronic acid (MeHA). In some embodiments, aphoto-crosslinkable polymer precursor comprises polyethylene (glycol)diacrylate (PEGDA), gelatin-methacryloyl (GelMA), methacrylatedhyaluronic acid (MeHA), or a combination thereof. In some embodiments, aphoto-crosslinkable polymer precursor (e.g., PAZAM) can be covalentlylinked (e.g., cross-linked) to a substrate. In some embodiments, aphoto-crosslinkable polymer precursor is not covalently linked to asubstrate surface. For example, a silane-free acrylamide can be used(See U.S. Pat. Application Publication No. 2011/0059865, hereinincorporated by reference in its entirety). The photo-crosslinkablepolymer precursor in a feature (e.g., droplet or bead) can bepolymerized by any known method. The oligonucleotides can be polymerizedin a cross-linked gel matrix (e.g., copolymerized or simultaneouslypolymerized). In some embodiments, the features containing thephoto-crosslinkable polymer precursor deposited on the substrate surfacecan be exposed to UV light. The UV light can induce polymerization ofthe photo-crosslinkable polymer precursor and result in the featuresbecoming a gel matrix (e.g., gel pads) on the substrate surface (e.g.,array).

Polymerization methods for hydrogel subunits can be selected to formhydrogels with different properties (e.g., pore volume, swellingproperties, biodegradability, conduction, transparency, and/orpermeability of the hydrogel). For example, a hydrogel can include poresof sufficient volume to allow the passage of macromolecules, (e.g.,nucleic acids, proteins, chromatin, metabolites, gRNA, antibodies,carbohydrates, peptides, metabolites, and/or small molecules) to/fromthe sample (e.g., tissue section). It is known that pore volumegenerally decreases with increasing concentration of hydrogel subunitsand generally increases with an increasing ratio of hydrogel subunits tocross-linker. Therefore, a hydrogel composition can be prepared thatincludes a concentration of hydrogel subunits that allows the passage ofsuch biological macromolecules.

In some embodiments, hydrogel formation on a substrate occurs before,contemporaneously with, or after features (e.g., beads) are attached tothe substrate. For example, when a capture probe is attached (e.g.,directly or indirectly) to a substrate, hydrogel formation can beperformed on the substrate already containing the capture probes.

(D) Arrays

In many of the methods described herein, features (as described furtherbelow) are collectively positioned on a substrate. An “array” is aspecific arrangement of a plurality of features that is either irregularor forms a regular pattern. Individual features in the array differ fromone another based on their relative spatial locations. In general, atleast two of the plurality of features in the array include a distinctcapture probe (e.g., any of the examples of capture probes describedherein).

Arrays can be used to measure large numbers of analytes simultaneously.In some embodiments, oligonucleotides are used, at least in part, tocreate an array. For example, one or more copies of a single species ofoligonucleotide (e.g., capture probe) can correspond to or be directlyor indirectly attached to a given feature in the array. In someembodiments, a given feature in the array includes two or more speciesof oligonucleotides (e.g., capture probes). In some embodiments, the twoor more species of oligonucleotides (e.g., capture probes) attacheddirectly or indirectly to a given feature on the array include a common(e.g., identical) spatial barcode.

(I) Arrays for Analyte Capture

In some embodiments, an array can include a capture probe attacheddirectly or indirectly to the substrate. The capture probe can include acapture domain (e.g., a nucleotide sequence) that can specifically bind(e.g., hybridize) to a target analyte (e.g., mRNA, DNA, or protein)within a sample. In some embodiments, the binding of the capture probeto the target (e.g., hybridization) can be detected and quantified bydetection of a visual signal, e.g., a fluorophore, a heavy metal (e.g.,silver ion), or chemiluminescent label, which has been incorporated intothe target. In some embodiments, the intensity of the visual signalcorrelates with the relative abundance of each analyte in the biologicalsample. Since an array can contain thousands or millions of captureprobes (or more), an array can interrogate many analytes in parallel.

In some embodiments, a substrate includes one or more capture probesthat are designed to capture analytes from one or more organisms. In anon-limiting example, a substrate can contain one or more capture probesdesigned to capture mRNA from one organism (e.g., a human) and one ormore capture probes designed to capture DNA from a second organism(e.g., a bacterium).

The capture probes can be attached to a substrate or feature using avariety of techniques. In some embodiments, the capture probe isdirectly attached to a feature that is fixed on an array. In someembodiments, the capture probes are immobilized to a substrate bychemical immobilization. For example, a chemical immobilization can takeplace between functional groups on the substrate and correspondingfunctional elements on the capture probes. Exemplary correspondingfunctional elements in the capture probes can either be an inherentchemical group of the capture probe, e.g., a hydroxyl group, or afunctional element can be introduced on to the capture probe. An exampleof a functional group on the substrate is an amine group. In someembodiments, the capture probe to be immobilized includes a functionalamine group or is chemically modified in order to include a functionalamine group. Means and methods for such a chemical modification are wellknown in the art.

In some embodiments, the capture probe is a nucleic acid. In someembodiments, the capture probe is immobilized on a substrate or featurevia its 5′ end. In some embodiments, the capture probe is immobilized ona substrate or feature via its 5′ end and includes from the 5′ to 3′end: one or more barcodes (e.g., a spatial barcode and/or a UMI) and oneor more capture domains. In some embodiments, the capture probe isimmobilized on a substrate or feature via its 5′ end and includes fromthe 5′ to 3′ end: one barcode (e.g., a spatial barcode or a UMI) and onecapture domain. In some embodiments, the capture probe is immobilized ona substrate or feature via its 5′ end and includes from the 5′ to 3′end: a cleavage domain, a functional domain, one or more barcodes (e.g.,a spatial barcode and/or a UMI), and a capture domain.

In some embodiments, the capture probe is immobilized on a substrate orfeature via its 5′ end and includes from the 5′ to 3′ end: a cleavagedomain, a functional domain, one or more barcodes (e.g., a spatialbarcode and/or a UMI), a second functional domain, and a capture domain.In some embodiments, the capture probe is immobilized on a substrate orfeature via its 5′ end and includes from the 5′ to 3′ end: a cleavagedomain, a functional domain, a spatial barcode, a UMI, and a capturedomain. In some embodiments, the capture probe is immobilized on asubstrate or feature via its 5′ end and does not include a spatialbarcode. In some embodiments, the capture probe is immobilized on asubstrate or feature via its 5′ end and does not include a UMI. In someembodiments, the capture probe includes a sequence for initiating asequencing reaction.

In some embodiments, the capture probe is immobilized on a substrate orfeature via its 3′ end. In some embodiments, the capture probe isimmobilized on a substrate or feature via its 3′ end and includes fromthe 3′ to 5′ end: one or more barcodes (e.g., a spatial barcode and/or aUMI) and one or more capture domains. In some embodiments, the captureprobe is immobilized on a substrate or feature via its 3′ end andincludes from the 3′ to 5′ end: one barcode (e.g., a spatial barcode ora UMI) and one capture domain. In some embodiments, the capture probe isimmobilized on a substrate or feature via its 3′ end and includes fromthe 3′ to 5′ end: a cleavage domain, a functional domain, one or morebarcodes (e.g., a spatial barcode and/or a UMI), and a capture domain.In some embodiments, the capture probe is immobilized on a substrate orfeature via its 3′ end and includes from the 3′ to 5′ end: a cleavagedomain, a functional domain, a spatial barcode, a UMI, and a capturedomain.

The localization of the functional group within the capture probe to beimmobilized can be used to control and shape the binding behavior and/ororientation of the capture probe, e.g., the functional group can beplaced at the 5′ or 3′ end of the capture probe or within the sequenceof the capture probe. In some embodiments, a capture probe can furtherinclude a substrate. A typical substrate for a capture probe to beimmobilized includes moieties which are capable of binding to suchcapture probes, e.g., to amine-functionalized nucleic acids. Examples ofsuch substrates are carboxy, aldehyde, or epoxy substrates.

In some embodiments, the substrates on which capture probes can beimmobilized can be chemically activated, e.g., by the activation offunctional groups available on the substrate. The term “activatedsubstrate” relates to a material in which interacting or reactivechemical functional groups are established or enabled by chemicalmodification procedures. For example, a substrate including carboxylgroups can be activated before use. Furthermore, certain substratescontain functional groups that can react with specific moieties alreadypresent in the capture probes.

In some embodiments, a covalent linkage is used to directly couple acapture probe to a substrate. In some embodiments a capture probe isindirectly coupled to a substrate through a linker separating the“first” nucleotide of the capture probe from the substrate, e.g., achemical linker. In some embodiments, a capture probe does not binddirectly to the substrate, but interacts indirectly, for example bybinding to a molecule which itself binds directly or indirectly to thesubstrate. In some embodiments, the capture probe is indirectly attachedto a substrate (e.g., attached to a substrate via a solution including apolymer).

In some embodiments where the capture probe is immobilized on a featureof the array indirectly, e.g., via hybridization to a surface probecapable of binding the capture probe, the capture probe can furtherinclude an upstream sequence (5′ to the sequence that hybridizes to thenucleic acid, e.g., RNA of the tissue sample) that is capable ofhybridizing to 5′ end of a surface probe. Alone, the capture domain ofthe capture probe can be seen as a capture domain oligonucleotide, whichcan be used in the synthesis of the capture probe in embodiments wherethe capture probe is immobilized on the array indirectly.

In some embodiments, a substrate is comprised of an inert material ormatrix (e.g., glass slides) that has been functionalized by, forexample, treating the substrate with a material comprising reactivegroups which enable immobilization of capture probes. See, for example,WO 2017/019456, the entire contents of which is herein incorporated byreference. Non-limiting examples include polyacrylamide hydrogelssupported on an inert substrate (e.g., glass slide; see WO 2005/065814and U.S. Pat. Application No. 2008/0280773, the entire contents of whichis incorporated herein by reference).

In some embodiments, functionalized biomolecules (e.g., capture probes)are immobilized on a functionalized substrate using covalent methods.Methods for covalent attachment include, for example, condensation ofamines and activated carboxylic esters (e.g., N-hydroxysuccinimideesters); condensation of amine and aldehydes under reductive aminationconditions; and cycloaddition reactions such as the Diels-Alder [4+2]reaction, 1,3-dipolar cycloaddition reactions, and [2+2] cycloadditionreactions. Methods for covalent attachment also include, for example,click chemistry reactions, including [3+2] cycloaddition reactions(e.g., Huisgen 1,3-dipolar cycloaddition reaction andcopper(I)-catalyzed azide-alkyne cycloaddition (CuAAC)); thiol-enereactions; the Diels-Alder reaction and inverse electron demandDiels-Alder reaction; [4+1] cycloaddition of isonitriles and tetrazines;and nucleophilic ring-opening of small carbocycles (e.g., epoxideopening with amino oligonucleotides). Methods for covalent attachmentalso include, for example, maleimides and thiols; and para-nitrophenylester-functionalized oligonucleotides and polylysine-functionalizedsubstrate. Methods for covalent attachment also include, for example,disulfide reactions; radical reactions (see, e.g., U.S. Pat. No.5,919,626, the entire contents of which are herein incorporated byreference); and hydrazide-functionalized substrate (e.g., wherein thehydrazide functional group is directly or indirectly attached to thesubstrate) and aldehyde-functionalized oligonucleotides (see, e.g.,Yershov et al. (1996) Proc. Natl. Acad. Sci. USA 93, 4913-4918, theentire contents of which are herein incorporated by reference).

In some embodiments, functionalized biomolecules (e.g., capture probes)are immobilized on a functionalized substrate using photochemicalcovalent methods. Methods for photochemical covalent attachment include,for example, immobilization of antraquinone-conjugated oligonucleotides(see, e.g., Koch et al. (2000) Bioconjugate Chem. 11, 474-483, theentire contents of which is herein incorporated by reference).

In some embodiments, functionalized biomolecules (e.g., capture probes)are immobilized on a functionalized substrate using non-covalentmethods. Methods for non-covalent attachment include, for example,biotin-functionalized oligonucleotides and streptavidin-treatedsubstrates (see, e.g., Holmstrøm et al. (1993) Analytical Biochemistry209, 278-283 and Gilles et al. (1999) Nature Biotechnology 17, 365-370,the entire contents of which are herein incorporated by reference).

In some embodiments, an oligonucleotide (e.g., a capture probe) can beattached to a substrate or feature according to the methods set forth inU.S. Pat. Nos. 6,737,236, 7,259,258, 7,375,234, 7,427,678, 5,610,287,5,807,522, 5,837,860, and 5,472,881; U.S. Pat. Application PublicationNos. 2008/0280773 and 2011/0059865; Shalon et al. (1996) GenomeResearch, 639-645; Rogers et al. (1999) Analytical Biochemistry 266,23-30; Stimpson et al. (1995) Proc. Natl. Acad. Sci. USA 92, 6379-6383;Beattie et al. (1995) Clin. Chem. 45, 700-706; Lamture et al. (1994)Nucleic Acids Research 22, 2121-2125; Beier et al. (1999) Nucleic AcidsResearch 27, 1970-1977; Joos et al. (1997) Analytical Biochemistry 247,96-101; Nikiforov et al. (1995) Analytical Biochemistry 227, 201-209;Timofeev et al. (1996) Nucleic Acids Research 24, 3142-3148; Chrisey etal. (1996) Nucleic Acids Research 24, 3031-3039; Guo et al. (1994)Nucleic Acids Research 22, 5456-5465; Running and Urdea (1990)BioTechniques 8, 276-279; Fahy et al. (1993) Nucleic Acids Research 21,1819-1826; Zhang et al. (1991) 19, 3929-3933; and Rogers et al. (1997)Gene Therapy 4, 1387-1392. The entire contents of each of the foregoingdocuments is incorporated herein by reference.

(II) Generation of Capture Probes in an Array Format

Arrays can be prepared by a variety of methods. In some embodiments,arrays are prepared through the synthesis (e.g., in situ synthesis) ofoligonucleotides on the array, or by jet printing or lithography. Forexample, light-directed synthesis of high-density DNA oligonucleotidescan be achieved by photolithography or solid-phase DNA synthesis. Toimplement photolithographic synthesis, synthetic linkers modified withphotochemical protecting groups can be attached to a substrate and thephotochemical protecting groups can be modified using aphotolithographic mask (applied to specific areas of the substrate) andlight, thereby producing an array having localized photo-deprotection.Many of these methods are known in the art, and are described e.g., inMiller et al., “Basic concepts of microarrays and potential applicationsin clinical microbiology.” Clinical Microbiology Reviews 22.4 (2009):611-633; US201314111482A; US9593365B2; US2019203275; and WO2018091676,which are each incorporated herein by reference in its entirety.

Spotting or Printing

In some embodiments, oligonucleotides (e.g., capture probes) can be“spotted” or “printed” onto a substrate to form an array. Theoligonucleotides can be applied by either noncontact or contactprinting. A noncontact printer can use the same method as computerprinters (e.g., bubble jet or inkjet) to expel small droplets of probesolution onto the substrate. The specialized inkjet-like printer canexpel nanoliter to picoliter volume droplets of oligonucleotidesolution, instead of ink, onto the substrate. In contact printing, eachprint pin directly applies the oligonucleotide solution onto a specificlocation on the surface. The oligonucleotides can be attached to thesubstrate surface by the electrostatic interaction of the negativecharge of the phosphate backbone of the DNA with a positively chargedcoating of the substrate surface or by UV-cross-linked covalent bondsbetween the thymidine bases in the DNA and amine groups on the treatedsubstrate surface. In some embodiments, the substrate is a glass slide.In some embodiments, the oligonucleotides (e.g., capture probes) areattached to a substrate by a covalent bond to a chemical matrix, e.g.,epoxy-silane, amino-silane, lysine, polyacrylamide, etc.

In Situ Synthesis

Capture probes arrays can be prepared by in situ synthesis. In someembodiments, capture probe arrays can be prepared usingphotolithography. Photolithography typically relies on UV masking andlight-directed combinatorial chemical synthesis on a substrate toselectively synthesize probes directly on the surface of an array, onenucleotide at a time per spot, for many spots simultaneously. In someembodiments, a substrate contains covalent linker molecules that have aprotecting group on the free end that can be removed by light. UV lightis directed through a photolithographic mask to deprotect and activateselected sites with hydroxyl groups that initiate coupling with incomingprotected nucleotides that attach to the activated sites. The mask isdesigned in such a way that the exposure sites can be selected, and thusspecify the coordinates on the array where each nucleotide can beattached. The process can be repeated, a new mask is applied activatingdifferent sets of sites and coupling different bases, allowing differentoligonucleotides to be constructed at each site. This process can beused to synthesize hundreds of thousands of different oligonucleotides.In some embodiments, maskless array synthesizer technology can be used.Programmable micromirrors can create digital masks that reflect thedesired pattern of UV light to deprotect the features.

In some embodiments, the inkjet spotting process can also be used for insitu oligonucleotide synthesis. The different nucleotide precursors pluscatalyst can be printed on the substrate, and are then combined withcoupling and deprotection steps. This method relies on printingpicoliter volumes of nucleotides on the array surface in repeated roundsof base-by-base printing that extends the length of the oligonucleotideprobes on the array.

Electric Fields

Arrays can also be prepared by active hybridization via electric fieldsto control nucleic acid transport. Negatively charged nucleic acids canbe transported to specific sites, or features, when a positive currentis applied to one or more test sites on the array. The surface of thearray can contain a binding molecule, e.g., streptavidin, which allowsfor the formation of bonds (e.g., streptavidin-biotin bonds) onceelectrically addressed biotinylated probes reach their targetedlocation. The positive current is then removed from the active features,and new test sites can be activated by the targeted application of apositive current. The process are repeated until all sites on the arrayare covered.

Ligation

In some embodiments, an array comprising barcoded probes can begenerated through ligation of a plurality of oligonucleotides. In someinstances, an oligonucleotide of the plurality contains a portion of abarcode, and the complete barcode is generated upon ligation of theplurality of oligonucleotides. For example, a first oligonucleotidecontaining a first portion of a barcode can be attached to a substrate(e.g., using any of the methods of attaching an oligonucleotide to asubstrate described herein), and a second oligonucleotide containing asecond portion of the barcode can then be ligated onto the firstoligonucleotide to generate a complete barcode. Different combinationsof the first, second and any additional portions of a barcode can beused to increase the diversity of the barcodes. In instances where thesecond oligonucleotide is also attached to the substrate prior toligation, the first and/or the second oligonucleotide can be attached tothe substrate via a surface linker which contains a cleavage site. Uponligation, the ligated oligonucleotide can be linearized by cleaving atthe cleavage site.

To increase the diversity of the barcodes, a plurality of secondoligonucleotides comprising two or more different barcode sequences canbe ligated onto a plurality of first oligonucleotides that comprise thesame barcode sequence, thereby generating two or more different speciesof barcodes. To achieve selective ligation, a first oligonucleotideattached to a substrate containing a first portion of a barcode caninitially be protected with a protective group (e.g., a photocleavableprotective group), and the protective group can be removed prior toligation between the first and second oligonucleotide. In instanceswhere the barcoded probes on an array are generated through ligation oftwo or more oligonucleotides, a concentration gradient of theoligonucleotides can be applied to a substrate such that differentcombinations of the oligonucleotides are incorporated into a barcodedprobe depending on its location on the substrate.

Probes can be generated by directly ligating additional oligonucleotidesonto existing oligonucleotides via a splint oligonucleotide. In someembodiments, oligonucleotides on an existing array can include arecognition sequence that can hybridize with a splint oligonucleotide.The recognition sequence can be at the free 5′ end or the free 3′ end ofan oligonucleotide on the existing array. Recognition sequences usefulfor the methods of the present disclosure may not contain restrictionenzyme recognition sites or secondary structures (e.g., hairpins), andmay include high contents of Guanine and Cytosine nucleotides.

Polymerases

Barcoded probes on an array can also be generated by adding singlenucleotides to existing oligonucleotides on an array, for example, usingpolymerases that function in a template-independent manner. Singlenucleotides can be added to existing oligonucleotides in a concentrationgradient, thereby generating probes with varying length, depending onthe location of the probes on the array.

Modification of Existing Capture Probes

Arrays can also be prepared by modifying existing arrays, for example,by modifying oligonucleotides already attached to an arrays. Forinstance, capture probes can be generated on an array that alreadycomprises oligonucleotides that are attached to the array (or featureson the array) at the 3′ end and have a free 5′ end. In some instances,an array is any commercially available array (e.g., any of the arraysavailable commercially as described herein). The oligonucleotides can bein situ synthesized using any of the in situ synthesis methods describedherein. The oligonucleotide can include a barcode and one or moreconstant sequences. In some instances, the constant sequences arecleavable sequences. The length of the oligonucleotides attached to thesubstrate (e.g., array) can be less than 100 nucleotides (e.g., lessthan 90, 80, 75, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, or 10nucleotides). To generate probes using oligonucleotides, a primercomplementary to a portion of an oligonucleotide (e.g., a constantsequence shared by the oligonucleotides) can hybridize to theoligonucleotide and extend the oligonucleotide (using theoligonucleotide as a template) to form a duplex and to create a 3′overhang. The 3′ overhang can be created by template-independent ligases(e.g., terminal deoxynucleotidyl transferase (TdT) or poly(A)polymerase). The 3′ overhang allows additional nucleotides oroligonucleotides to be added to the duplex, for example, by an enzyme.For instance, a capture probe can be generated by adding additionaloligonucleotides to the end of the 3′ overhang (e.g., via splintoligonucleotide mediated ligation), where the additionaloligonucleotides can include a sequence or a portion of sequence of oneor more capture domains, or a complement thereof.

The additional oligonucleotide (e.g., a sequence or a portion ofsequence of a capture domain) can include a degenerate sequence (e.g.,any of the degenerate sequences as described herein). The additionaloligonucleotide (e.g., a sequence or a portion of sequence of a capturedomain) can include a sequence compatible for hybridizing or ligatingwith an analyte of interest in a biological sample. An analyte ofinterest can also be used as a splint oligonucleotide to ligateadditional oligonucleotides onto a probe. When using a splintoligonucleotide to assist in the ligation of additionaloligonucleotides, an additional oligonucleotide can include a sequencethat is complementary to the sequence of the splint oligonucleotide.Ligation of the oligonucleotides can involve the use of an enzyme, suchas, but not limited to, a ligase. Non-limiting examples of suitableligases include Tth DNA ligase, Taq DNA ligase, Thermococcus sp. (strain9oN) DNA ligase (9oNTM DNA ligase, New England Biolabs), AmpligaseTM(available from Epicentre Biotechnologies, Madison, WI), and SplintR(available from New England Biolabs, Ipswich, MA). An array generated asdescribed above is useful for spatial analysis of a biological sample.For example, the one or more capture domains can be used to hybridizewith the poly(A) tail of an mRNA molecule. Reverse transcription can becarried out using a reverse transcriptase to generate cDNA complementaryto the captured mRNA. The sequence and location of the captured mRNA canthen be determined (e.g., by sequencing the capture probe that containsthe barcode as well as the complementary cDNA).

An array for spatial analysis can be generated by various methods asdescribed herein. In some embodiments, the array has a plurality ofcapture probes comprising spatial barcodes. These spatial barcodes andtheir relationship to the locations on the array can be determined. Insome cases, such information is readily available, because theoligonucleotides are spotted, printed, or synthesized on the array witha pre-determined pattern. In some cases, the spatial barcode can bedecoded by methods described herein, e.g., by in situ sequencing, byvarious labels associated with the spatial barcodes etc. In someembodiments, an array can be used as a template to generate a daughterarray. Thus, the spatial barcode can be transferred to the daughterarray with a known pattern.

(III) Features

A “feature” is an entity that acts as a support or repository forvarious molecular entities used in sample analysis. In some embodiments,some or all of the features in an array are functionalized for analytecapture. In some embodiments, functionalized features include one ormore capture probe(s). Examples of features include, but are not limitedto, a bead, a spot of any two- or three-dimensional geometry (e.g., anink jet spot, a masked spot, a square on a grid), a well, and a hydrogelpad. In some embodiments, features are directly or indirectly attachedor fixed to a substrate. In some embodiments, the features are notdirectly or indirectly attached or fixed to a substrate, but instead,for example, are disposed within an enclosed or partially enclosed threedimensional space (e.g., wells or divots).

In addition to those above, a wide variety of other features can be usedto form the arrays described herein. For example, in some embodiments,features that are formed from polymers and/or biopolymers that are jetprinted, screen printed, or electrostatically deposited on a substratecan be used to form arrays. Jet printing of biopolymers is described,for example, in PCT Pat. Application Publication No. WO 2014/085725. Jetprinting of polymers is described, for example, in de Gans et al., AdvMater. 16(3): 203-213 (2004). Methods for electrostatic deposition ofpolymers and biopolymers are described, for example, in Hoyer et al.,Anal. Chem. 68(21): 3840-3844 (1996). The entire contents of each of theforegoing references are incorporated herein by reference.

As another example, in some embodiments, features are formed by metallicmicro- or nanoparticles. Suitable methods for depositing such particlesto form arrays are described, for example, in Lee et al., Beilstein J.Nanotechnol. 8: 1049-1055 (2017), the entire contents of which areincorporated herein by reference.

As a further example, in some embodiments, features are formed bymagnetic particles that are assembled on a substrate. Examples of suchparticles and methods for assembling arrays are described in Ye et al.,Scientific Reports 6: 23145 (2016), the entire contents of which areincorporated herein by reference.

As another example, in some embodiments, features correspond to regionsof a substrate in which one or more optical labels have beenincorporated, and/or which have been altered by a process such aspermanent photobleaching. Suitable substrates to implement features inthis manner include a wide variety of polymers, for example. Methods forforming such features are described, for example, in Moshrefzadeh etal., Appl. Phys. Lett. 62: 16 (1993), the entire contents of which areincorporated herein by reference.

As yet another example, in some embodiments, features can correspond tocolloidal particles assembled (e.g., via self-assembly) to form anarray. Suitable colloidal particles are described for example in Sharma,Resonance 23(3): 263-275 (2018), the entire contents of which areincorporated herein by reference.

As a further example, in some embodiments, features can be formed viaspot-array photopolymerization of a monomer solution on a substrate. Inparticular, two-photon and three-photon polymerization can be used tofabricate features of relatively small (e.g., submicron) dimensions.Suitable methods for preparing features on a substrate in this mannerare described for example in Nguyen et al., Materials Today 20(6):314-322 (2017), the entire contents of which are incorporated herein byreference.

In some embodiments, features are directly or indirectly attached orfixed to a substrate that is liquid permeable. In some embodiments,features are directly or indirectly attached or fixed to a substratethat is biocompatible. In some embodiments, features are directly orindirectly attached or fixed to a substrate that is a hydrogel.

FIG. 12 depicts an exemplary arrangement of barcoded features within anarray. From left to right, FIG. 12 shows (L) a slide including sixspatially-barcoded arrays, (C) an enlarged schematic of one of the sixspatially-barcoded arrays, showing a grid of barcoded features inrelation to a biological sample, and (R) an enlarged schematic of onesection of an array, showing the specific identification of multiplefeatures within the array (labelled as ID578, ID579, ID560, etc.).

Beads

A “bead” can be a particle. A bead can be porous, non-porous, solid,semi-solid, and/or a combination thereof. In some embodiments, a beadcan be dissolvable, disruptable, and/or degradable, whereas in certainembodiments, a bead is not degradable. A semi-solid bead can be aliposomal bead. Solid beads can include metals including, withoutlimitation, iron oxide, gold, and silver. In some embodiments, the beadcan be a silica bead. In some embodiments, the bead can be rigid. Insome embodiments, the bead can be flexible and/or compressible.

The bead can be a macromolecule. The bead can be formed of nucleic acidmolecules bound together. The bead can be formed via covalent ornon-covalent assembly of molecules (e.g., macromolecules), such asmonomers or polymers. Polymers or monomers can be natural or synthetic.Polymers or monomers can be or include, for example, nucleic acidmolecules (e.g., DNA or RNA).

A bead can be rigid, or flexible and/or compressible. A bead can includea coating including one or more polymers. Such a coating can bedisruptable or dissolvable. In some embodiments, a bead includes aspectral or optical label (e.g., dye) attached directly or indirectly(e.g., through a linker) to the bead. For example, a bead can beprepared as a colored preparation (e.g., a bead exhibiting a distinctcolor within the visible spectrum) that can change color (e.g.,colorimetric beads) upon application of a desired stimulus (e.g., heatand/or chemical reaction) to form differently colored beads (e.g.,opaque and/or clear beads).

A bead can include natural and/or synthetic materials. For example, abead can include a natural polymer, a synthetic polymer or both naturaland synthetic polymers. Examples of natural polymers include, withoutlimitation, proteins, sugars such as deoxyribonucleic acid, rubber,cellulose, starch (e.g., amylose, amylopectin), enzymes,polysaccharides, silks, polyhydroxyalkanoates, chitosan, dextran,collagen, carrageenan, ispaghula, acacia, agar, gelatin, shellac,sterculia gum, xanthan gum, corn sugar gum, guar gum, gum karaya,agarose, alginic acid, alginate, or natural polymers thereof. Examplesof synthetic polymers include, without limitation, acrylics, nylons,silicones, spandex, viscose rayon, polycarboxylic acids, polyvinylacetate, polyacrylamide, polyacrylate, polyethylene glycol,polyurethanes, polylactic acid, silica, polystyrene, polyacrylonitrile,polybutadiene, polycarbonate, polyethylene, polyethylene terephthalate,poly(chlorotrifluoroethylene), poly(ethylene oxide), poly(ethyleneterephthalate), polyethylene, polyisobutylene, poly(methylmethacrylate), poly(oxymethylene), polyformaldehyde, polypropylene,polystyrene, poly(tetrafluoroethylene), poly(vinyl acetate), poly(vinylalcohol), poly(vinyl chloride), poly(vinylidene dichloride),poly(vinylidene difluoride), poly(vinyl fluoride) and/or combinations(e.g., co-polymers) thereof. Beads can also be formed from materialsother than polymers, including for example, lipids, micelles, ceramics,glass-ceramics, material composites, metals, and/or other inorganicmaterials.

In some embodiments, a bead is a degradable bead. A degradable bead caninclude one or more species (e.g., disulfide linkers, primers, otheroligonucleotides, etc.) with a labile bond such that, when thebead/species is exposed to the appropriate stimuli, the labile bond isbroken and the bead degrades. The labile bond can be a chemical bond(e.g., covalent bond, ionic bond) or can be another type of physicalinteraction (e.g., van der Waals interactions, dipole-dipoleinteractions, etc.). In some embodiments, a cross-linker used togenerate a bead can include a labile bond. Upon exposure to theappropriate conditions, the labile bond can be broken and the beaddegraded. For example, upon exposure of a polyacrylamide gel beadincluding cystamine cross-linkers to a reducing agent, the disulfidebonds of the cystamine can be broken and the bead degraded.

Degradation can refer to the disassociation of a bound or entrainedspecies (e.g., disulfide linkers, primers, other oligonucleotides, etc.)from a bead, both with and without structurally degrading the physicalbead itself. For example, entrained species can be released from beadsthrough osmotic pressure differences due to, for example, changingchemical environments. By way of example, alteration of bead porevolumes due to osmotic pressure differences can generally occur withoutstructural degradation of the bead itself. In some embodiments, anincrease in pore volume due to osmotic swelling of a bead can permit therelease of entrained species within the bead. In some embodiments,osmotic shrinking of a bead can cause a bead to better retain anentrained species due to pore volume contraction.

Any suitable agent that can degrade beads can be used. In someembodiments, changes in temperature or pH can be used to degradethermo-sensitive or pH-sensitive bonds within beads. In someembodiments, chemical degrading agents can be used to degrade chemicalbonds within beads by oxidation, reduction or other chemical changes.For example, a chemical degrading agent can be a reducing agent, such asDTT, where DTT can degrade the disulfide bonds formed between across-linker and gel precursors, thus degrading the bead. In someembodiments, a reducing agent can be added to degrade the bead, whichcan cause the bead to release its contents. Examples of reducing agentscan include, without limitation, dithiothreitol (DTT),β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamineor DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinationsthereof.

Any of a variety of chemical agents can be used to trigger thedegradation of beads. Examples of chemical agents include, but are notlimited to, pH-mediated changes to the integrity of a component withinthe bead, degradation of a component of a bead via cleavage ofcross-linked bonds, and depolymerization of a component of a bead.

In some embodiments, a bead can be formed from materials that includedegradable chemical cross-linkers, such as N,N′-bis-(acryloyl)cystamine(BAC) or cystamine. Degradation of such degradable cross-linkers can beaccomplished through any variety of mechanisms. In some examples, a beadcan be contacted with a chemical degrading agent that can induceoxidation, reduction or other chemical changes. For example, a chemicaldegrading agent can be a reducing agent, such as dithiothreitol (DTT).Additional examples of reducing agents can include β-mercaptoethanol,(2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA),tris(2-carboxyethyl) phosphine (TCEP), or combinations thereof.

In some embodiments, exposure to an aqueous solution, such as water, cantrigger hydrolytic degradation, and thus degradation of the bead. Beadscan also be induced to release their contents upon the application of athermal stimulus. A change in temperature can cause a variety of changesto a bead. For example, heat can cause a solid bead to liquefy. A changein heat can cause melting of a bead such that a portion of the beaddegrades. In some embodiments, heat can increase the internal pressureof the bead components such that the bead ruptures or explodes. Heat canalso act upon heat-sensitive polymers used as materials to constructbeads.

Where degradable beads are used, it can be beneficial to avoid exposingsuch beads to the stimulus or stimuli that cause such degradation priorto a given time, in order to, for example, avoid premature beaddegradation and issues that arise from such degradation, including forexample poor flow characteristics and aggregation. By way of example,where beads include reducible cross-linking groups, such as disulfidegroups, it will be desirable to avoid contacting such beads withreducing agents, e.g., DTT or other disulfide cleaving reagents. In suchembodiments, treatment of the beads described herein will, in someembodiments be provided free of reducing agents, such as DTT. Becausereducing agents are often provided in commercial enzyme preparations, itcan be desirable to provide reducing agent free (or DTT free) enzymepreparations in treating the beads described herein. Examples of suchenzymes include, e.g., polymerase enzyme preparations, reversetranscriptase enzyme preparations, ligase enzyme preparations, as wellas many other enzyme preparations that can be used to treat the beadsdescribed herein. The terms “reducing agent free” or “DTT free”preparations refer to a preparation having less than about ⅒, less thanabout 1/50th, or less than about 1/100th of the lower ranges for suchmaterials used in degrading the beads. For example, for DTT, thereducing agent free preparation can have less than about 0.01 millimolar(mM), 0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or less than about 0.0001mM DTT. In some embodiments, the amount of DTT can be undetectable.

A degradable bead can be useful to more quickly release an attachedcapture probe (e.g., a nucleic acid molecule, a spatial barcodesequence, and/or a primer) from the bead when the appropriate stimulusis applied to the bead as compared to a bead that does not degrade. Forexample, for a species bound to an inner surface of a porous bead or inthe case of an encapsulated species, the species can have greatermobility and accessibility to other species in solution upon degradationof the bead. In some embodiments, a species can also be attached to adegradable bead via a degradable linker (e.g., disulfide linker). Thedegradable linker can respond to the same stimuli as the degradable beador the two degradable species can respond to different stimuli. Forexample, a capture probe having one or more spatial barcodes can beattached, via a disulfide bond, to a polyacrylamide bead includingcystamine. Upon exposure of the spatially-barcoded bead to a reducingagent, the bead degrades and the capture probe having the one or morespatial barcode sequences is released upon breakage of both thedisulfide linkage between the capture probe and the bead and thedisulfide linkages of the cystamine in the bead.

The addition of multiple types of labile bonds to a bead can result inthe generation of a bead capable of responding to varied stimuli. Eachtype of labile bond can be sensitive to an associated stimulus (e.g.,chemical stimulus, light, temperature, pH, enzymes, etc.) such thatrelease of reagents attached to a bead via each labile bond can becontrolled by the application of the appropriate stimulus. Somenon-limiting examples of labile bonds that can be coupled to a precursoror bead include an ester linkage (e.g., cleavable with an acid, a base,or hydroxylamine), a vicinal diol linkage (e.g., cleavable via sodiumperiodate), a Diels-Alder linkage (e.g., cleavable via heat), a sulfonelinkage (e.g., cleavable via a base), a silyl ether linkage (e.g.,cleavable via an acid), a glycosidic linkage (e.g., cleavable via anamylase), a peptide linkage (e.g., cleavable via a protease), or aphosphodiester linkage (e.g., cleavable via a nuclease (e.g., DNAase)).A bond can be cleavable via other nucleic acid molecule targetingenzymes, such as restriction enzymes (e.g., restriction endonucleases).Such functionality can be useful in controlled release of reagents froma bead. In some embodiments, another reagent including a labile bond canbe linked to a bead after gel bead formation via, for example, anactivated functional group of the bead as described above. In someembodiments, a gel bead including a labile bond is reversible. In someembodiments, a gel bead with a reversible labile bond is used to captureone or more regions of interest of a biological sample. For example,without limitation, a bead including a thermolabile bond can be heatedby a light source (e.g., a laser) that causes a change in the gel beadthat facilitates capture of a biological sample in contact with the gelbead. Capture probes having one or more spatial barcodes that arereleasably, cleavably, or reversibly attached to the beads describedherein include capture probes that are released or releasable throughcleavage of a linkage between the capture probe and the bead, or thatare released through degradation of the underlying bead itself, allowingthe capture probes having the one or more spatial barcodes to beaccessed or become accessible by other reagents, or both.

Beads can have different physical properties. Physical properties ofbeads can be used to characterize the beads. Non-limiting examples ofphysical properties of beads that can differ include volume, shape,circularity, density, symmetry, and hardness. For example, beads can beof different volumes. Beads of different diameters can be obtained byusing microfluidic channel networks configured to provide beads of aspecific volume (e.g., based on channel sizes, flow rates, etc.). Insome embodiments, beads have different hardness values that can beobtained by varying the concentration of polymer used to generate thebeads. In some embodiments, a spatial barcode attached to a bead can bemade optically detectable using a physical property of the captureprobe. For example, a nucleic acid origami, such as a deoxyribonucleicacid (DNA) origami, can be used to generate an optically detectablespatial barcode. To do so, a nucleic acid molecule, or a plurality ofnucleic acid molecules, can be folded to create two-and/orthree-dimensional geometric shapes. The different geometric shapes canbe optically detected.

In some embodiments, special types of nanoparticles with more than onedistinct physical property can be used to make the beads physicallydistinguishable. For example, Janus particles with both hydrophilic andhydrophobic surfaces can be used to provide unique physical properties.

A bead can generally be of any suitable shape. Examples of bead shapesinclude, but are not limited to, spherical, non-spherical, oval, oblong,amorphous, circular, cylindrical, cuboidal, hexagonal, and variationsthereof. In some embodiments, non-spherical (e.g., hexagonal, cuboidal,shaped beads can assemble more closely (e.g., tighter) than sphericalshaped beads. In some embodiments, beads can self-assemble into amonolayer. A cross section (e.g., a first cross-section) can correspondto a diameter or maximum cross-sectional dimension of the bead. In someembodiments, the bead can be approximately spherical. In suchembodiments, the first cross-section can correspond to the diameter ofthe bead. In some embodiments, the bead can be approximatelycylindrical. In such embodiments, the first cross-section can correspondto a diameter, length, or width along the approximately cylindricalbead.

Beads can be of uniform size or heterogeneous size. “Polydispersity”generally refers to heterogeneity of sizes of molecules or particles.The polydispersity index (PDI) of a bead can be calculated using theequation PDI = Mw/Mn, where Mw is the weight-average molar mass and Mnis the number-average molar mass. In certain embodiments, beads can beprovided as a population or plurality of beads having a relativelymonodisperse size distribution. Where it can be desirable to providerelatively consistent amounts of reagents, maintaining relativelyconsistent bead characteristics, such as size, can contribute to theoverall consistency.

In some embodiments, the beads provided herein can have sizedistributions that have a coefficient of variation in theircross-sectional dimensions of less than 50%, less than 40%, less than30%, less than 20%, less than 15%, less than 10%, less than 5%, orlower. In some embodiments, a plurality of beads provided herein has apolydispersity index of less than 50%, less than 45%, less than 40%,less than 35%, less than 30%, less than 25%, less than 20%, less than15%, less than 10%, less than 5%, or lower.

In some embodiments, the bead can have a diameter or maximum dimensionno larger than 100 µm (e.g., no larger than 95 µm, 90 µm, 85 µm, 80 µm,75 µm, 70 µm, 65 µm, 60 µm, 55 µm, 50 µm, 45 µm, 40 µm, 35 µm, 30 µm, 25µm, 20 µm, 15 µm, 14 µm, 13 µm, 12 µm, 11 µm, 10 µm, 9 µm, 8 µm, 7 µm, 6µm, 5 µm, 4 µm, 3 µm, 2 µm, or 1 µm.)

In some embodiments, a plurality of beads has an average diameter nolarger than 100 µm. In some embodiments, a plurality of beads has anaverage diameter or maximum dimension no larger than 95 µm, 90 µm, 85µm, 80 µm, 75 µm, 70 µm, 65 µm, 60 µm, 55 µm, 50 µm, 45 µm, 40 µm, 35µm, 30 µm, 25 µm, 20 µm, 15 µm, 14 µm, 13 µm, 12 µm, 11 µm, 10 µm, 9 µm,8 µm, 7 µm, 6 µm, 5 µm, 4 µm, 3 µm, 2 µm, or 1 µm.

In some embodiments, the volume of the bead can be at least about 1 µm³,e.g., at least 1 µm³, 2 µm³, 3 µm^(3,) 4 µm³, 5 µm³, 6 µm³, 7 µm³, 8µm³, 9 µm³, 10 µm³, 12 µm³, 14 µm³, 16 µm³, 18 µm^(3,) 20 µm³, 25 µm³,30 µm³, 35 µm^(3,) 40 µm³, 45 µm³, 50 µm³, 55 µm^(3,) 60 µm³, 65 µm^(3,)70 µm^(3,) 75 µm³, 80 µm³, 85 µm³, 90 µm³, 95 µm³, 100 µm³, 125 µm³, 150µm³, 175 µm³, 300 µm³, 350 µm³, 400 µm³, 450 µm³, µm³, 500 µm³, 550 µm³,600 µm³, 650 µm³, 700 µm³, 750 µm³, 800 µm³, 850 µm³, 900 µm³, 950 µm³,1000 µm³, 1200 µm³, 1400 µm³, 1600 µm³, 1800 µm³, 2000 µm³, 2200 µm³,2400 µm³, 2600 µm³, 2800 µm³, 3000 µm³, or greater.

In some embodiments, the bead can have a volume of between about 1 µm³and 100 µm³, such as between about 1 µm³ and 10 µm³, between about10 µm³and 50 µm³, or between about 50 µm³ and 100 µm³. In some embodiments,the bead can include a volume of between about 100 µm³ and 1000 µm³,such as between about 100 µm³ and 500 µm³ or between about 500 µm³ and1000 µm³. In some embodiments, the bead can include a volume betweenabout 1000 µm³ and 3000 µm³, such as between about 1000 µm³ and 2000 µm³or between about 2000 µm³ and 3000 µm³. In some embodiments, the beadcan include a volume between about 1 µm³ and 3000 µm³, such as betweenabout 1 µm³ and 2000 µm³, between about 1 µm³ and 1000 µm³, betweenabout 1 µm³ and 500 µm³, or between about 1 µm³ and 250 µm³.

The bead can include one or more cross-sections that can be the same ordifferent. In some embodiments, the bead can have a first cross-sectionthat is different from a second cross-section. The bead can have a firstcross-section that is at least about 0.0001 micrometer, 0.001micrometer, 0.01 micrometer, 0.1 micrometer, or 1 micrometer. In someembodiments, the bead can include a cross-section (e.g., a firstcross-section) of at least about 1 micrometer (µm), 2 µm, 3 µm, 4 µm, 5µm, 6 µm, 7 µm, 8 µm, 9 µm, 10 µm, 11 µm, 12 µm, 13 µm, 14 µm, 15 µm, 16µm, 17 µm, 18 µm, 19 µm, 20 µm, 25 µm, 30 µm, 35 µm, 40 µm, 45 µm, 50µm, 55 µm, 60 µm, 65 µm, 70 µm, 75 µm, 80 µm, 85 µm, 90 µm, 100 µm, 120µm, 140 µm, 160 µm, 180 µm, 200 µm, 250 µm, 300 µm, 350 µm, 400 µm, 450µm, 500 µm, 550 µm, 600 µm, 650 µm, 700 µm, 750 µm, 800 µm, 850 µm, 900µm, 950 µm, 1 millimeter (mm), or greater. In some embodiments, the beadcan include a cross-section (e.g., a first cross-section) of betweenabout 1 µm and 500 µm, such as between about 1 µm and 100 µm, betweenabout 100 µm and 200 µm, between about 200 µm and 300 µm, between about300 µm and 400 µm, or between about 400 µm and 500 µm. For example, thebead can include a cross-section (e.g., a first cross-section) ofbetween about 1 µm and 100 µm. In some embodiments, the bead can have asecond cross-section that is at least about 1 µm. For example, the beadcan include a second cross-section of at least about 1 micrometer (µm),2 µm, 3 µm, 4 µm, 5 µm, 6 µm, 7 µm, 8 µm, 9 µm, 10 µm, 11 µm, 12 µm, 13µm, 14 µm, 15 µm, 16 µm, 17 µm, 18 µm, 19 µm, 20 µm, 25 µm, 30 µm, 35µm, 40 µm, 45 µm, 50 µm, 55 µm, 60 µm, 65 µm, 70 µm, 75 µm, 80 µm, 85µm, 90 µm, 100 µm, 120 µm, 140 µm, 160 µm, 180 µm, 200 µm, 250 µm, 300µm, 350 µm, 400 µm, 450 µm, 500 µm, 550 µm, 600 µm, 650 µm, 700 µm, 750µm, 800 µm, 850 µm, 900 µm, 950 µm, 1 millimeter (mm), or greater. Insome embodiments, the bead can include a second cross-section of betweenabout 1 µm and 500 µm, such as between about 1 µm and 100 µm, betweenabout 100 µm and 200 µm, between about 200 µm and 300 µm, between about300 µm and 400 µm, or between about 400 µm and 500 µm. For example, thebead can include a second cross-section of between about 1 µm and 100µm.

In some embodiments, beads can be of a nanometer scale (e.g., beads canhave a diameter or maximum cross-sectional dimension of about 100nanometers (nm) to about 900 nanometers (nm) (e.g., 850 nm or less, 800nm or less, 750 nm or less, 700 nm or less, 650 nm or less, 600 nm orless, 550 nm or less, 500 nm or less, 450 nm or less, 400 nm or less,350 nm or less, 300 nm or less, 250 nm or less, 200 nm or less, 150 nmor less). A plurality of beads can have an average diameter or averagemaximum cross-sectional dimension of about 100 nanometers (nm) to about900 nanometers (nm) (e.g., 850 nm or less, 800 nm or less, 750 nm orless, 700 nm or less, 650 nm or less, 600 nm or less, 550 nm or less,500 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nmor less, 250 nm or less, 200 nm or less, 150 nm or less). In someembodiments, a bead has a diameter or volume that is about the diameterof a single cell (e.g., a single cell under evaluation).

In some embodiments, a bead is able to identify multiple analytes (e.g.,nucleic acids, proteins, chromatin, metabolites, drugs, gRNA, andlipids) from a single cell. In some embodiments, a bead is able toidentify a single analyte from a single cell (e.g., mRNA).

A bead can have a tunable pore volume. The pore volume can be chosen to,for instance, retain denatured nucleic acids. The pore volume can bechosen to maintain diffusive permeability to exogenous chemicals such assodium hydroxide (NaOH) and/or endogenous chemicals such as inhibitors.A bead can be formed of a biocompatible and/or biochemically compatiblematerial, and/or a material that maintains or enhances cell viability. Abead can be formed from a material that can be depolymerized thermally,chemically, enzymatically, and/or optically.

In some embodiments, beads can be non-covalently loaded with one or morereagents. The beads can be non-covalently loaded by, for instance,subjecting the beads to conditions sufficient to swell the beads,allowing sufficient time for the reagents to diffuse into the interiorsof the beads, and subjecting the beads to conditions sufficient tode-swell the beads. Swelling of the beads can be accomplished, forinstance, by placing the beads in a thermodynamically favorable solvent,subjecting the beads to a higher or lower temperature, subjecting thebeads to a higher or lower ion concentration, and/or subjecting thebeads to an electric field.

The swelling of the beads can be accomplished by various swellingmethods. In some embodiments, swelling is reversible (e.g., bysubjecting beads to conditions that promote de-swelling). In someembodiments, the de-swelling of the beads is accomplished, for instance,by transferring the beads in a thermodynamically unfavorable solvent,subjecting the beads to lower or higher temperatures, subjecting thebeads to a lower or higher ion concentration, and/or adding or removingan electric field. The de-swelling of the beads can be accomplished byvarious de-swelling methods. In some embodiments, de-swelling isreversible (e.g., subject beads to conditions that promote swelling). Insome embodiments, the de-swelling of beads can include transferring thebeads to cause pores in the bead to shrink. The shrinking can thenhinder reagents within the beads from diffusing out of the interiors ofthe beads. The hindrance created can be due to steric interactionsbetween the reagents and the interiors of the beads. The transfer can beaccomplished microfluidically. For instance, the transfer can beachieved by moving the beads from one co-flowing solvent stream to adifferent co-flowing solvent stream. The swellability and/or pore volumeof the beads can be adjusted by changing the polymer composition of thebead.

A bead can include a polymer that is responsive to temperature so thatwhen the bead is heated or cooled, the characteristics or dimensions ofthe bead can change. For example, a polymer can includepoly(N-isopropylacrylamide). A gel bead can includepoly(N-isopropylacrylamide) and when heated the gel bead can decrease inone or more dimensions (e.g., a cross-sectional diameter, multiplecross-sectional diameters). A temperature sufficient for changing one ormore characteristics of the gel bead can be, for example, at least about0° C. (°C), 1° C., 2° C., 3° C., 4° C., 5° C., 10° C., or higher. Forexample, the temperature can be about 4° C. In some embodiments, atemperature sufficient for changing one or more characteristics of thegel bead can be, for example, at least about 25° C., 30° C., 35° C., 37°C., 40° C., 45° C., 50° C., or higher. For example, the temperature canbe about 37° C.

Functionalization of beads for attachment of capture probes can beachieved through a wide range of different approaches, including,without limitation, activation of chemical groups within a polymer,incorporation of active or activatable functional groups in the polymerstructure, or attachment at the pre-polymer or monomer stage in beadproduction. The bead can be functionalized to bind to targeted analytes,such as nucleic acids, proteins, carbohydrates, lipids, metabolites,peptides, or other analytes.

In some embodiments, a bead can contain molecular precursors (e.g.,monomers or polymers), which can form a polymer network viapolymerization of the molecular precursors. In some embodiments, aprecursor can be an already polymerized species capable of undergoingfurther polymerization via, for example, a chemical cross-linkage. Insome embodiments, a precursor can include one or more of an acrylamideor a methacrylamide monomer, oligomer, or polymer. In some embodiments,the bead can include prepolymers, which are oligomers capable of furtherpolymerization. For example, polyurethane beads can be prepared usingprepolymers. In some embodiments, a bead can contain individual polymersthat can be further polymerized together (e.g., to form a co-polymer).In some embodiments, a bead can be generated via polymerization ofdifferent precursors, such that they include mixed polymers,co-polymers, and/or block co-polymers. In some embodiments, a bead caninclude covalent or ionic bonds between polymeric precursors (e.g.,monomers, oligomers, and linear polymers), nucleic acid molecules (e.g.,oligonucleotides), primers, and other entities. In some embodiments,covalent bonds can be carbon-carbon bonds or thioether bonds.

Cross-linking of polymers can be permanent or reversible, depending uponthe particular cross-linker used. Reversible cross-linking can allow thepolymer to linearize or dissociate under appropriate conditions. In someembodiments, reversible cross-linking can also allow for reversibleattachment of a material bound to the surface of a bead. In someembodiments, a cross-linker can form a disulfide linkage. In someembodiments, a chemical cross-linker forming a disulfide linkage can becystamine or a modified cystamine.

For example, where the polymer precursor material includes a linearpolymer material, such as a linear polyacrylamide, PEG, or other linearpolymeric material, the activation agent can include a cross-linkingagent, or a chemical that activates a cross-linking agent within formeddroplets. Likewise, for polymer precursors that include polymerizablemonomers, the activation agent can include a polymerization initiator.For example, in certain embodiments, where the polymer precursorincludes a mixture of acrylamide monomer with aN,N′-bis-(acryloyl)cystamine (BAC) comonomer, an agent such astetraethylmethylenediamine (TEMED) can be provided, which can initiatethe copolymerization of the acrylamide and BAC into a cross-linkedpolymer network, or other conditions sufficient to polymerize or gel theprecursors. The conditions sufficient to polymerize or gel theprecursors can include exposure to heating, cooling, electromagneticradiation, and/or light.

Following polymerization or gelling, a polymer or gel can be formed. Thepolymer or gel can be diffusively permeable to chemical or biochemicalreagents. The polymer or gel can be diffusively impermeable tomacromolecular constituents. The polymer or gel can include one or moreof disulfide cross-linked polyacrylamide, agarose, alginate, polyvinylalcohol, polyethylene glycol (PEG)-diacrylate, PEG-acrylate, PEG-thiol,PEG-azide, PEG-alkyne, other acrylates, chitosan, hyaluronic acid,collagen, fibrin, gelatin, or elastin. The polymer or gel can includeany other polymer or gel.

In some embodiments, disulfide linkages can be formed between molecularprecursor units (e.g., monomers, oligomers, or linear polymers) orprecursors incorporated into a bead and nucleic acid molecules (e.g.,oligonucleotides, capture probes). Cystamine (including modifiedcystamines), for example, is an organic agent including a disulfide bondthat can be used as a cross-linker agent between individual monomeric orpolymeric precursors of a bead. Polyacrylamide can be polymerized in thepresence of cystamine or a species including cystamine (e.g., a modifiedcystamine) to generate polyacrylamide gel beads including disulfidelinkages (e.g., chemically degradable beads includingchemically-reducible cross-linkers). The disulfide linkages can permitthe bead to be degraded (or dissolved) upon exposure of the bead to areducing agent.

In some embodiments, chitosan, a linear polysaccharide polymer, can becross-linked with glutaraldehyde via hydrophilic chains to form a bead.Crosslinking of chitosan polymers can be achieved by chemical reactionsthat are initiated by heat, pressure, change in pH, and/or radiation.

In some embodiments, a bead can include an acrydite moiety, which incertain aspects can be used to attach one or more capture probes to thebead. In some embodiments, an acrydite moiety can refer to an acryditeanalogue generated from the reaction of acrydite with one or morespecies (e.g., disulfide linkers, primers, other oligonucleotides,etc.), such as, without limitation, the reaction of acrydite with othermonomers and cross-linkers during a polymerization reaction. Acryditemoieties can be modified to form chemical bonds with a species to beattached, such as a capture probe. Acrydite moieties can be modifiedwith thiol groups capable of forming a disulfide bond or can be modifiedwith groups already including a disulfide bond. The thiol or disulfide(via disulfide exchange) can be used as an anchor point for a species tobe attached or another part of the acrydite moiety can be used forattachment. In some embodiments, attachment can be reversible, such thatwhen the disulfide bond is broken (e.g., in the presence of a reducingagent), the attached species is released from the bead. In someembodiments, an acrydite moiety can include a reactive hydroxyl groupthat can be used for attachment of species.

In some embodiments, precursors (e.g., monomers or cross-linkers) thatare polymerized to form a bead can include acrydite moieties, such thatwhen a bead is generated, the bead also includes acrydite moieties. Theacrydite moieties can be attached to a nucleic acid molecule (e.g., anoligonucleotide), which can include a priming sequence (e.g., a primerfor amplifying target nucleic acids, random primer, primer sequence formessenger RNA) and/or one or more capture probes. The one or morecapture probes can include sequences that are the same for all captureprobes coupled to a given bead and/or sequences that are differentacross all capture probes coupled to the given bead. The capture probecan be incorporated into the bead. In some embodiments, the captureprobe can be incorporated or attached to the bead such that the captureprobe retains a free 3′ end. In some embodiments, the capture probe canbe incorporated or attached to the bead such that the capture proberetains a free 5′ end. In some embodiments, beads can be functionalizedsuch that each bead contains a plurality of different capture probes.For example, a bead can include a plurality of capture probes e.g.,Capture Probe 1, Capture Probe 2, and Capture Probe 3, and each ofCapture Probes 1, Capture Probes 2, and Capture Probes 3 contain adistinct capture domain (e.g., capture domain of Capture Probe 1includes a poly(dT) capture domain, capture domain of Capture Probe 2includes a gene-specific capture domain, and capture domain of CaptureProbe 3 includes a CRISPR-specific capture domain). By functionalizingbeads to contain a plurality of different capture domains per bead, thelevel of multiplex capability for analyte detection can be improved.

In some embodiments, precursors (e.g., monomers or cross-linkers) thatare polymerized to form a bead can include a functional group that isreactive or capable of being activated such that when it becomesreactive it can be polymerized with other precursors to generate beadsincluding the activated or activatable functional group. The functionalgroup can then be used to attach additional species (e.g., disulfidelinkers, primers, other oligonucleotides, etc.) to the beads. Forexample, some precursors including a carboxylic acid (COOH) group canco-polymerize with other precursors to form a bead that also includes aCOOH functional group. In some embodiments, acrylic acid (a speciesincluding free COOH groups), acrylamide, and bis(acryloyl)cystamine canbe co-polymerized together to generate a bead including free COOHgroups. The COOH groups of the bead can be activated (e.g., via1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-Hydroxysuccinimide (NHS) or4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM)) such that they are reactive (e.g., reactive to amine functionalgroups where EDC/NHS or DMTMM are used for activation). The activatedCOOH groups can then react with an appropriate species (e.g., a speciesincluding an amine functional group where the carboxylic acid groups areactivated to be reactive with an amine functional group) as a functionalgroup on a moiety to be linked to the bead.

Beads including disulfide linkages in their polymeric network can befunctionalized with additional species (e.g., disulfide linkers,primers, other oligonucleotides, etc.) via reduction of some of thedisulfide linkages to free thiols. The disulfide linkages can be reducedvia, for example, the action of a reducing agent (e.g., DTT, TCEP, etc.)to generate free thiol groups, without dissolution of the bead. Freethiols of the beads can then react with free thiols of a species or aspecies including another disulfide bond (e.g., via thiol-disulfideexchange) such that the species can be linked to the beads (e.g., via agenerated disulfide bond). In some embodiments, free thiols of the beadscan react with any other suitable group. For example, free thiols of thebeads can react with species including an acrydite moiety. The freethiol groups of the beads can react with the acrydite via Michaeladdition chemistry, such that the species including the acrydite islinked to the bead. In some embodiments, uncontrolled reactions can beprevented by inclusion of a thiol capping agent such asN-ethylmalieamide or iodoacetate.

Activation of disulfide linkages within a bead can be controlled suchthat only a small number of disulfide linkages are activated. Controlcan be exerted, for example, by controlling the concentration of areducing agent used to generate free thiol groups and/or concentrationof reagents used to form disulfide bonds in bead polymerization. In someembodiments, a low concentration of reducing agent (e.g., molecules ofreducing agent:gel bead ratios) of less than or equal to about1:100,000,000,000, less than or equal to about 1:10,000,000,000, lessthan or equal to about 1:1,000,000,000, less than or equal to about1:100,000,000, less than or equal to about 1:10,000,000, less than orequal to about 1:1,000,000, less than or equal to about 1:100,000, orless than or equal to about 1:10,000) can be used for reduction.Controlling the number of disulfide linkages that are reduced to freethiols can be useful in ensuring bead structural integrity duringfunctionalization. In some embodiments, optically-active agents, such asfluorescent dyes can be coupled to beads via free thiol groups of thebeads and used to quantify the number of free thiols present in a beadand/or track a bead.

In some embodiments, addition of moieties to a bead after bead formationcan be advantageous. For example, addition of a capture probe after beadformation can avoid loss of the species (e.g., disulfide linkers,primers, other oligonucleotides, etc.) during chain transfer terminationthat can occur during polymerization. In some embodiments, smallerprecursors (e.g., monomers or cross linkers that do not include sidechain groups and linked moieties) can be used for polymerization and canbe minimally hindered from growing chain ends due to viscous effects. Insome embodiments, functionalization after bead synthesis can minimizeexposure of species (e.g., oligonucleotides) to be loaded withpotentially damaging agents (e.g., free radicals) and/or chemicalenvironments. In some embodiments, the generated hydrogel can possess anupper critical solution temperature (UCST) that can permit temperaturedriven swelling and collapse of a bead. Such functionality can aid inoligonucleotide (e.g., a primer) infiltration into the bead duringsubsequent functionalization of the bead with the oligonucleotide.Post-production functionalization can also be useful in controllingloading ratios of species in beads, such that, for example, thevariability in loading ratio is minimized. Species loading can also beperformed in a batch process such that a plurality of beads can befunctionalized with the species in a single batch.

Reagents can be encapsulated in beads during bead generation (e.g.,during polymerization of precursors). Such reagents can or cannotparticipate in polymerization. Such reagents can be entered intopolymerization reaction mixtures such that generated beads include thereagents upon bead formation. In some embodiments, such reagents can beadded to the beads after formation. Such reagents can include, forexample, capture probes (e.g., oligonucleotides), reagents for a nucleicacid amplification reaction (e.g., primers, polymerases, dNTPs,co-factors (e.g., ionic co-factors), buffers) including those describedherein, reagents for enzymatic reactions (e.g., enzymes, co-factors,chemical substrates, buffers), reagents for nucleic acid modificationreactions such as polymerization, ligation, or digestion, and/orreagents for template preparation (e.g., tagmentation) for one or moresequencing platforms (e.g., Nextera® for Illumina®). Such reagents caninclude one or more enzymes described herein, including withoutlimitation, polymerase, reverse transcriptase, restriction enzymes(e.g., endonuclease), transposase, ligase, proteinase K, DNAse, etc.Such reagents can also or alternatively include one or more reagentssuch as lysis agents, inhibitors, inactivating agents, chelating agents,stimulus agents. Trapping of such reagents can be controlled by thepolymer network density generated during polymerization of precursors,control of ionic charge within the bead (e.g., via ionic species linkedto polymerized species), or by the release of other species.Encapsulated reagents can be released from a bead upon bead degradationand/or by application of a stimulus capable of releasing the reagentsfrom the bead. In some embodiments, the beads or bead arrangements canbe incubated in permeabilization reagents as described herein.

In some embodiments, the beads can also include (e.g., encapsulate orhave attached thereto) a plurality of capture probes that includespatial barcodes, and the optical properties of the spatial barcodes canbe used for optical detection of the beads. For example, the absorbanceof light by the spatial barcodes can be used to distinguish the beadsfrom one another. In some embodiments, a detectable label can directlyor indirectly attach to a spatial barcode and provide optical detectionof the bead. In some embodiments, each bead in a group of one or morebeads has a unique detectable label, and detection of the uniquedetectable label determines the location of the spatial barcode sequenceassociated with the bead.

Optical properties giving rise to optical detection of beads can be dueto optical properties of the bead surface (e.g., a detectable labelattached to the bead), or optical properties from the bulk region of thebead (e.g., a detectable label incorporated during bead formation or anoptical property of the bead itself). In some embodiments, a detectablelabel can be associated with a bead or one or more moieties coupled tothe bead.

In some embodiments, the beads include a plurality of detectable labels.For example, a fluorescent dye can be attached to the surface of thebeads and/or can be incorporated into the beads. Different intensitiesof the different fluorescent dyes can be used to increase the number ofoptical combinations that can be used to differentiate between beads.For example, if N is the number of fluorescent dyes (e.g., between 2 and10 fluorescent dyes, such as 4 fluorescent dyes) and M is the possibleintensities for the dyes (e.g., between 2 and 50 intensities, such as 20intensities), then M^(N) are the possible distinct optical combinations.In one example, 4 fluorescent dyes with 20 possible intensities can beused to generate 160,000 distinct optical combinations.

One or more optical properties of the beads or biological contents, suchas cells or nuclei, can be used to distinguish the individual beads orbiological contents from other beads or biological contents. In someembodiments, the beads are made optically detectable by including adetectable label having optical properties to distinguish the beads fromone another.

In some embodiments, optical properties of the beads can be used foroptical detection of the beads. For example, without limitation, opticalproperties can include absorbance, birefringence, color, fluorescence,luminosity, photosensitivity, reflectivity, refractive index,scattering, or transmittance. For example, beads can have differentbirefringence values based on degree of polymerization, chain length, ormonomer chemistry.

In some embodiments, nanobeads, such as quantum dots or Janus beads, canbe used as optical labels or components thereof. For example, a quantumdot can be attached to a spatial barcode of a bead.

Optical labels of beads can provide enhanced spectral resolution todistinguish (e.g., identify) between beads with unique spatial barcodes(e.g., beads including unique spatial barcode sequences). That is, thebeads are manufactured in a way that the optical labels and the barcodeson the beads (e.g., spatial barcodes) are correlated with each other. Insome aspects, the beads can be loaded into a flowcell such that beadsare arrayed in a closely packed manner (e.g., single-cell resolution).Imaging can be performed, and the spatial location of the barcodes canbe determined (e.g., based on information from a look-up table (LUT)).The optical labels for spatial profiling allow for quick deconvolutionof bead-barcode (e.g., spatial barcode) identify.

In some examples, a lookup table (LUT) can be used to associate aproperty (e.g., an optical label, such as a color and/or intensity) ofthe bead with the barcode sequence. The property may derive from theparticle (e.g., bead) or an optical label associated with the bead. Thebeads can be imaged to obtain optical information of the bead,including, for example, the property (e.g., color and/or intensity) ofthe bead or the optical label associated with the bead, and opticalinformation of the biological sample. For example, an image can includeoptical information in the visible spectrum, non-visible spectrum, orboth. In some embodiments, multiple images can be obtained acrossvarious optical frequencies.

In some embodiments, a first bead includes a first optical label andspatial barcodes each having a first spatial barcode sequence. A secondbead includes a second optical label and spatial barcodes each having asecond spatial barcode sequence. The first optical label and secondoptical label can be different (e.g., provided by two differentfluorescent dyes or the same fluorescent dye at two differentintensities). The first and second spatial barcode sequences can bedifferent nucleic acid sequences. In some embodiments, the beads can beimaged to identify the first and second optical labels, and the firstand second optical labels can then be used to associate the first andsecond optical labels with the first and second spatial barcodesequences, respectively. In some embodiments, the nucleic acidcontaining the spatial barcode can further have a capture domain foranalytes (e.g., mRNA). In some embodiments, the nucleic acid (e.g.,nucleic acid containing the spatial barcode) can have a unique molecularidentifier, a cleavage domain, a functional domain, or combinationsthereof.

In some embodiments, the optical label has a characteristicelectromagnetic spectrum. As used herein, the “electromagnetic spectrum”refers to the range of frequencies of electromagnetic radiation. In someembodiments, the optical label has a characteristic absorption spectrum.As used herein, the “absorption spectrum” refers to the range offrequencies of electromagnetic radiation that are absorbed. The“electromagnetic spectrum” or “absorption spectrum” can lead todifferent characteristic spectrum. In some embodiments, the peakradiation or the peak absorption occurs at 380-450 nm (Violet), 450-485nm (Blue), 485-500 nm (Cyan), 500-565 nm (Green), 565-590 nm (Yellow),590-625 nm (Orange), or 625-740 nm (Red). In some embodiments, the peakradiation or the peak absorption occurs around 400 nm, 460 nm, or 520nm.

Optical labels included on the beads can identify the associated spatialbarcode on the bead. Due to the relative limited diversity of opticallabels it can be advantageous to limit the size of the spatial array fordeconvolution. For example, the substrate can be partitioned into two ormore partitions (e.g., bins). In some embodiments, the substrate can bepartitioned into three or more partitions. In some embodiments, thesubstrate can be partitioned into four or more partitions (e.g., bins).In some embodiments, a set of beads are deposited to the partition.Within each set of beads, one or more beads (e.g., equal to or more than10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400,450, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, or 5000 beads) canhave an unique optical label.

In some cases, beads within the same partition can have differentcoordinates on the substrate. These coordinates can be determined e.g.,by various imaging techniques, such as observation through microscopeunder an appropriate condition. In some embodiments, the beads withinthe same partition can share the same spatial barcode. In someembodiments, the beads (e.g., beads having capture probes with barcodes,e.g., spatial barcodes or UMI) are different from each other fordifferent partition bins. In some embodiments, the beads having captureprobes with barcodes (e.g., spatial barcodes or UMI) can have differentbarcodes. For example, in some cases, within each set of beads, whichbeads are associated with a capture probe, the capture probes onindividual beads can have a unique barcode. In some cases, among allbeads (e.g., within two or more sets of beads), individual beads canhave capture probes with a unique barcode.

In some aspects, the present disclosure provides a substrate. Thesubstrate can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300,400, 500, 600, 700, 800, 900, 1000, or more than 1000 partitions (e.g.,bins, or pre-defined area). The partitions can have the same shape ordifferent shapes. In some embodiments, the substrate has only onepartition (e.g., bin or pre-defined area).

In some embodiments, the first partition (e.g., the first pre-definedarea, or the only bin on the substrate) can have a first set of beads.In some embodiments, at least one bead from the first set of beadscomprises an optical label, and a capture probe (e.g., anoligonucleotide capture probe) comprising a barcode and a capturedomain. At least one of the beads can have a unique optical label amongthe first set of beads. In some embodiments, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% ofthe beads in the first set of beads have a unique optical label. In someembodiments, each bead in the first set of beads has a unique opticallabel.

In some embodiments, the substrate can have a second partition (e.g.,the second pre-defined area, or the second bin). The second partitioncan have a second set of beads. In some embodiments, at least one beadfrom the second set of beads comprises an optical label, and a captureprobe (e.g., an oligonucleotide capture probe) comprising a barcode anda capture domain. At least one of the beads can have a unique opticallabel among the second set of beads. In some embodiments, at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,99.9% of the beads in the second set of beads have a unique opticallabel. In some embodiments, each bead in the second set of beads has aunique optical label.

In some embodiments, the substrate can have a third partition, a fourthpartition, a fifth partition, a sixth partition, a seventh partition, aneighth partition, a ninth partition, or a tenth partition, etc. In someembodiments, the substrate can have multiple partitions. In some cases,each of these partitions has properties that are similar to the first orthe second partitions described herein. For example, at least one beadfrom each set of beads comprises an optical label, and a capture probe(e.g., an oligonucleotide capture probe) comprising a barcode and acapture domain. At least one of these beads can have a unique opticallabel among each set of beads. In some embodiments, at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%of the beads in each set of beads have a unique optical label. In someembodiments, each bead in each set of beads has a unique optical label.

In some embodiments, the beads are deposited on the substrate. In someembodiments, the beads can be deposited directly on or into a biologicalsample. Thus, in some cases, the biological sample can be fixed orattached on the substrate before beads are deposited onto the substrate.

In some embodiments, the beads are only deposited to areas of interest(e.g., specific locations on the substrate, specific cell types, andspecific tissue structures). Thus, the deposited beads do notnecessarily cover the entire biological sample. In some embodiments, oneor more regions of a substrate can be masked or modified (e.g., cappedcapture domains) such that the masked regions do not interact with acorresponding region of the biological sample.

In some embodiments, two or more than two sets of beads (e.g., 2, 3, 4,5, 6, 7, 8, 9, 10, or more than 10 sets) are deposited at two or morethan two partitions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10partitions). These partitions do not need to be adjacent to each other.As long as the location of the partitions on the substrate is recorded,the identity of the beads can be determined from the optical labels.

In some embodiments, a set of beads can have equal to or more than 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200,250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 2000, 3000,4000, or 5000 beads. In some embodiments, a set 25 of beads can haveless than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150,200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 2000, 3000,4000, or 5000 beads.

Optical labels can be included while generating the beads. For example,optical labels can be included in the polymer structure of a gel bead,or attached at the pre-polymer or monomer stage in bead production. Insome embodiments, the beads include moieties that attach to one or moreoptical labels (e.g., at a surface of a bead and/or within a bead). Insome embodiments, optical labels can be loaded into the beads with oneor more reagents. For example, reagents and optical labels can be loadedinto the beads by diffusion of the reagents (e.g., a solution ofreagents including the optical labels). In some embodiments, opticallabels can be included while preparing spatial barcodes. For example,spatial barcodes can be prepared by synthesizing molecules includingbarcode sequences (e.g., using a split pool or combinatorial approach).Optical labels can be attached to spatial barcodes prior to attachingthe spatial barcodes to a bead. In some embodiments, optical labels canbe included after attaching spatial barcodes to a bead. For example,optical labels can be attached to spatial barcodes coupled to the bead.In some embodiments, spatial barcodes or sequences thereof can bereleasably or cleavably attached to the bead. Optical labels can bereleasably or non-releasably attached to the bead. In some embodiments,a first bead (e.g., a bead including a plurality of spatial barcodes)can be coupled to a second bead including one or more optical labels.For example, the first bead can be covalently coupled to the second beadvia a chemical bond. In some embodiments, the first bead can benon-covalently associated with the second bead.

The first and/or second bead can include a plurality of spatialbarcodes. The plurality of spatial barcodes coupled to a given bead caninclude the same barcode sequences. Where both the first and secondbeads include spatial barcodes, the first and second beads can includespatial barcodes including the same barcode sequences or differentbarcode sequences.

Bead arrays containing captured analytes can be processed in bulk orpartitioned into droplet emulsions for preparing sequencing libraries.In some embodiments, next generation sequencing reads are clustered andcorrelated to the spatial position of the spatial barcode on the beadarray. For example, the information can be computationally superimposedover a high-resolution image of the tissue section to identify thelocation(s), where the analytes were detected.

In some embodiments, de-cross linking can be performed to account forde-crosslinking chemistries that may be incompatible with certainbarcoding/library prep biochemistry (e.g., presence of proteases). Forexample, a two-step process is possible. In the first step, beads can beprovided in droplets such that DNA binds to the beads after theconventional de-crosslinking chemistry is performed. In the second step,the emulsion is broken and beads collected and then re-encapsulatedafter washing for further processing.

In some embodiments, beads can be affixed or attached to a substrateusing photochemical methods. For example, a bead can be functionalizedwith perfluorophenylazide silane (PFPA silane), contacted with asubstrate, and then exposed to irradiation (see, e.g., Liu et al. (2006)Journal of the American Chemical Society 128, 14067-14072). For example,immobilization of antraquinone-functionalized substrates (see, e.g.,Koch et al. (2000) Bioconjugate Chem. 11, 474-483, the entire contentsof which are herein incorporated by reference).

The arrays can also be prepared by bead self-assembly. Each bead can becovered with hundreds of thousands of copies of a specificoligonucleotide. In some embodiments, each bead can be covered withabout 1,000 to about 1,000,000 oligonucleotides. In some embodiments,each bead can be covered with about 1,000,000 to about 10,000,000oligonucleotides. In some embodiments, each bead can covered with about2,000,000 to about 3,000,000, about 3,000,000 to about 4,000,000, about4,000,000 to about 5,000,000, about 5,000,000 to about 6,000,000, about6,000,000 to about 7,000,000, about 7,000,000 to about 8,000,000, about8,000,000 to about 9,000,000, or about 9,000,000 to about 10,000,000oligonucleotides. In some embodiments, each bead can be covered withabout 10,000,000 to about 100,000,000 oligonucleotides. In someembodiments, each bead can be covered with about 100,000,000 to about1,000,000,000 oligonucleotides. In some embodiments, each bead can becovered with about 1,000,000,000 to about 10,000,000,000oligonucleotides. The beads can be irregularly distributed across etchedsubstrates during the array production process. During this process, thebeads can be self-assembled into arrays (e.g., on a fiber-optic bundlesubstrate or a silica slide substrate). In some embodiments, the beadsirregularly arrive at their final location on the array. Thus, the beadlocation may need to be mapped or the oligonucleotides may need to besynthesized based on a predetermined pattern.

Beads can be affixed or attached to a substrate covalently,non-covalently, with adhesive, or a combination thereof. The attachedbeads can be, for example, layered in a monolayer, a bilayer, atrilayer, or as a cluster. As defined herein, a “monolayer” generallyrefers to an arrayed series of probes, beads, spots, dots, features,micro-locations, or islands that are affixed or attached to a substrate,such that the beads are arranged as one layer of single beads. In someembodiments, the beads are closely packed.

As defined herein, the phrase “substantial monolayer” or “substantiallyform(s) a monolayer” generally refers to (the formation of) an arrayedseries of probes, beads, microspheres, spots, dots, features,micro-locations, or islands that are affixed or attached to a substrate,such that about 50% to about 99% (e.g., about 50% to about 98%) of thebeads are arranged as one layer of single beads. This arrangement can bedetermined using a variety of methods, including microscopic imaging.

In some embodiments, the monolayer of beads is a located in a predefinedarea on the substrate. For example, the predefined area can bepartitioned with physical barriers, a photomask, divots in thesubstrate, or wells in the substrate.

As used herein, the term “reactive element” generally refers to amolecule or molecular moiety that can react with another molecule ormolecular moiety to form a covalent bond. Reactive elements include, forexample, amines, aldehydes, alkynes, azides, thiols, haloacetyls,pyridyl disulfides, hydrazides, carboxylic acids, alkoxyamines,sulfhydryls, maleimides, Michael acceptors, hydroxyls, and activeesters. Some reactive elements, for example, carboxylic acids, can betreated with one or more activating agents (e.g., acylating agents,isourea-forming agents) to increase susceptibility of the reactiveelement to nucleophilic attack. Non-limiting examples of activatingagents include N-hydroxysuccinimide, N-hydroxysulfosuccinimide,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, dicyclohexylcarbodiimide,diisopropylcarbodiiimide, 1-hydroxybenzotriazole,(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexfluorophosphate,(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate,4-(N,N-dimethylamino)pyridine, and carbonyldiimidazole.

In some embodiments, the reactive element is bound directly to a bead.For example, hydrogel beads can be treated with an acrylic acid monomerto form acrylic acid-functionalized hydrogel beads. In some cases, thereactive element is bound indirectly to the bead via one or morelinkers. As used herein, a “linker” generally refers to amultifunctional (e.g., bifunctional, trifunctional) reagent used forconjugating two or more chemical moieties. A linker can be a cleavablelinker that can undergo induced dissociation. For example, thedissociation can be induced by a solvent (e.g., hydrolysis andsolvolysis); by irradiation (e.g., photolysis); by an enzyme (e.g.,enzymolysis); or by treatment with a solution of specific pH (e.g., pH4, 5, 6, 7, or 8).

In some embodiments, the reactive element is bound directly to asubstrate. For example, a glass slide can be coated with(3-aminopropyl)triethoxysilane. In some embodiments, the reactiveelement is bound indirectly to a substrate via one or more linkers.

Gel / Hydrogel Beads

In some embodiments, the bead can be a gel bead. A “gel” is a semi-rigidmaterial permeable to liquids and gases. Exemplary gels include, but arenot limited to, those having a colloidal structure, such as agarose;polymer mesh structures, such as gelatin; hydrogels; and cross-linkedpolymer structures, such as polyacrylamide, SFA (see, for example, U.S.Pat. Application Publication No. 2011/0059865, which is incorporatedherein by reference in its entirety) and PAZAM (see, for example, U.S.Pat. Application Publication No. 2014/0079923, which is incorporatedherein by reference in its entirety).

A gel can be formulated into various shapes and dimensions depending onthe context of intended use. In some embodiments, a gel is prepared andformulated as a gel bead (e.g., a gel bead including capture probesattached or associated with the gel bead). A gel bead can be a hydrogelbead. A hydrogel bead can be formed from molecular precursors, such as apolymeric or monomeric species.

In some cases, a bead comprises a polymer or hydrogel. The polymer orhydrogel may determine one or more characteristics of the hydrogel bead,such as the volume, fluidity, porosity, rigidity, organization, or oneor more other features of the hydrogel bead. In some embodiments, ahydrogel bead can include a polymer matrix (e.g., a matrix formed bypolymerization or cross-linking). A polymer matrix can include one ormore polymers (e.g., polymers having different functional groups orrepeat units). Cross-linking can be via covalent, ionic, and/orinductive interactions, and/or physical entanglement.

A polymer or hydrogel may be formed, for example, upon cross-linking oneor more cross-linkable molecules within the hydrogel bead. For example,a hydrogel may be formed upon cross-linking one or more molecules withinthe hydrogel bead. The hydrogel may be formed upon polymerizing aplurality of monomers within the hydrogel bead. The hydrogel may beformed upon polymerizing a plurality of polymers within the hydrogelbead. Polymeric or hydrogel precursors may be provided to the hydrogelbead and may not form a polymer or hydrogel without application of astimulus (e.g., as described herein). In some cases, the hydrogel beadmay be encapsulated within the polymer or hydrogel. Formation of ahydrogel bead may take place following one or more other changes to thecell that may be brought about by one or more other conditions.

The methods described herein may be applied to a single hydrogel bead ora plurality of hydrogel beads. A method of processing a plurality ofhydrogel beads may comprise providing the plurality of hydrogel beadswithin a vessel and subjecting the plurality of hydrogel beads toconditions sufficient to change one or more characteristics of thehydrogel bead. For example, plurality of hydrogel beads may be subjectedto a first condition or set of conditions comprising a chemical species,and a cross-section of the hydrogel beads of the plurality of hydrogelbeads may change from a first cross-section to a second cross-section,which second cross-section is less than the first cross-section. Thechemical species may comprise, for example, an organic solvent such asethanol, methanol, or acetone. The plurality of hydrogel beads may thenbe subjected to a second condition or set of conditions comprising achemical species, and crosslinks may form within each of the hydrogelbeads. The chemical species may comprise, for example, a cross-linkingagent. The plurality of processed hydrogel beads may be provided in anaqueous fluid. In some instances, the second cross-section of theplurality of hydrogel beads is substantially maintained in the aqueousfluid. The plurality of processed hydrogel beads may be partitionedwithin a plurality of partitions. The partitions may be, for example,aqueous droplets included in a water-in-oil emulsion. The partitions maybe, for example, a plurality of wells. The plurality of fixed hydrogelbeads may be co-partitioned with one or more reagents. In some cases,the plurality of fixed hydrogel beads may be co-partitioned with one ormore beads, where each bead comprises a plurality of nucleic acidbarcode molecules attached thereto. The nucleic acid barcode moleculesattached to a given bead may comprise a common barcode sequence, and thenucleic acid barcode molecules attached to each different bead maycomprise a sequence comprising a different common barcode sequence. Thenucleic acid barcode molecules, or portions thereof, may then be used inreactions with target molecules associated with hydrogel beads of theplurality of hydrogel beads.

Core/Shell Beads

In some embodiments, the bead is a core/shell bead that comprises aninner core (e.g., a nanosphere or microsphere) and an outer shell (e.g.,a hydrogel coating the nanosphere or microsphere). In some embodiments,the inner core can be a solid nanoparticle or solid microparticle. Insome embodiments, the inner core can be a silica inner core (e.g., asilica nanoparticle or silica microparticle). In some embodiments, theinner core of the core/shell bead can have an average diameter of about1 micron. In some embodiments, the inner core can have an averagediameter of about 2 microns. In some embodiments, the inner core canhave an average diameter of about 3 microns. In some embodiments, theinner core can have an average diameter of about 4 microns. In someembodiments, the inner core can have an average diameter of about 5microns. In some embodiments, the inner core can have an averagediameter of about 6 microns. In some embodiments, the inner core canhave an average diameter of about 7 microns. In some embodiments, theinner core can have an average diameter of about 8 microns. In someembodiments, the inner core can have an average diameter of about 9microns. In some embodiments, the inner core can have an averagediameter of about 10 microns. In some embodiments, the inner core canhave an average diameter of about 100 nanometers to about 10 microns.

In some embodiments, the core/shell bead can decrease its outer shellvolume by removing solvents, salts, or water (e.g., dehydrated,desiccated, dried, exsiccated) from the outer shell to form a shrunkencore/shell bead. In another example, the core/shell bead can decreaseits outer shell volume by adjusting temperature or pH, as describedabove. In some embodiments, the core/shell bead can expand its outershell volume, for example by the addition of solvents, salts, or water(e.g., rehydration) to form an expanded core/shell bead. In someembodiments, the outer shell (e.g., coating the inner core) can have anaverage thickness of about 1 micron. In some embodiments, the outershell can have an average thickness of about 2 microns. In someembodiments, the outer shell can have an average thickness of about 3microns. In some embodiments, the outer shell can have an averagethickness of about 4 microns. In some embodiments, the outer shell canhave an average thickness of about 5 microns.

In some embodiments, the core/shell bead can have an average diameter ofabout 1 micron to about 10 microns. In some embodiments, the core/shellbead can have an average diameter of about 1 micron. In someembodiments, the core/shell bead can have an average diameter of about 2microns. In some embodiments, the core/shell bead can have an averagediameter of about 3 microns. In some embodiments, the core/shell beadcan have an average diameter of about 4 microns. In some embodiments,the core/shell bead can have an average diameter of about 5 microns. Insome embodiments, the core/shell bead can have an average diameter ofabout 6 microns. In some embodiments, the core/shell bead can have anaverage diameter of about 7 microns. In some embodiments, the core/shellbead can have an average diameter of about 8 microns. In someembodiments, the core/shell bead can have an average diameter of about 9microns. In some embodiments, the core/shell bead can have an averagediameter of about 10 microns.

Methods for Covalently Bonding Features to a Substrate

Provided herein are methods for the covalent bonding of features (e.g.,optically labeled beads, hydrogel beads, microsphere beads) to asubstrate.

In some embodiments, the features (e.g., beads) are coupled to asubstrate via a covalent bond between a first reactive element and asecond reactive element. In some embodiments, the covalently-bound beadssubstantially form a monolayer of features (e.g., hydrogel beads,microsphere beads) on the substrate.

In some embodiments, the features (e.g., beads) are functionalized witha first reactive element, which is directly bound to the features. Insome embodiments, the features are functionalized with a first reactiveelement, which is indirectly bound to the beads via a linker. In someembodiments, the linker is a benzophenone. In some embodiments, thelinker is an amino methacrylamide. For example, the linker can be3-aminopropyl methacrylamide. In some embodiments, the linker is a PEGlinker. In some embodiments, the linker is a cleavable linker.

In some embodiments, the substrate is functionalized with a secondreactive element, which is directly bound to the substrate. In someembodiments, the substrate is functionalized with a second reactiveelement, which is indirectly bound to the beads via a linker. In someembodiments, the linker is a benzophenone. For example, the linker canbe benzophenone. In some embodiments, the linker is an aminomethacrylamide. For example, the linker can be 3-aminopropylmethacrylamide. In some embodiments, the linker is a PEG linker. In someembodiments, the linker is a cleavable linker.

In some embodiments, the substrate is a glass slide. In someembodiments, the substrate is a pre-functionalized glass slide.

In some embodiments, about 99% of the covalently-bound beads form amonolayer of beads on the substrate. In some embodiments, about 50% toabout 98% form a monolayer of beads on the substrate. For example, about50% to about 95%, about 50% to about 90%, about 50% to about 85%, about50% to about 80%, about 50% to about 75%, about 50% to about 70%, about50% to about 65%, about 50% to about 60%, or about 50% to about 55% ofthe covalently-bound beads form a monolayer of beads on the substrate.In some embodiments, about 55% to about 98%, about 60% to about 98%,about 65% to about 98%, about 70% to about 98%, about 75% to about 98%,about 80% to about 98%, about 85% to about 98%, about 90% to about 95%,or about 95% to about 98% of the covalently-bound beads form a monolayerof beads on the substrate. In some embodiments, about 55% to about 95%,about 60% to about 90%, about 65% to about 95%, about 70% to about 95%,about 75% to about 90%, about 75% to about 95%, about 80% to about 90%,about 80% to about 95%, about 85% to about 90%, or about 85% to about95% of the covalently-bound beads for a monolayer of beads on thesubstrate.

In some embodiments, at least one of the first reactive element and thesecond reactive element is selected from the group consisting of:

wherein

-   R¹ is selected from H, C₁-C₆ alkyl, or —SO₃;-   R² is C₁-C₆ alkyl; and-   X is a halo moiety.

In some embodiments, at least one of the first reactive element or thesecond reactive element comprises

wherein the

indicates the point of attachment of the first reactive element or thesecond reactive element to the bead (e.g., hydrogel bead or microspherebead) or to the substrate.

In some embodiments, at least one of the first reactive element or thesecond reactive element is selected from the group consisting of:

wherein

-   R¹ is selected from H, C₁-C₆ alkyl, or —SO₃;-   R² is C₁-C₆ alkyl; and-   X is a halo moiety.

In some embodiments, at least one of the first reactive element or thesecond reactive element comprises

wherein R¹ is selected from H, C₁-C₆ alkyl, or —SO₃. In someembodiments, R¹ is H. In some embodiments, R¹ is C₁-C₆ alkyl. In someembodiments, R¹ is —SO₃.

In some embodiments, at least one of the first reactive element or thesecond reactive element comprises

wherein R² is C₁-C₆ alkyl. In some embodiments, R² is methyl.

In some embodiments, at least one of the first reactive element or thesecond reactive element comprises

Is some embodiments,

can be reacted with an activating agent to form an active ester. In someembodiments, the active ester is

In some embodiments, the activating agent is an acylating agent (e.g.,N-hydroxysuccinimide and N-hydroxysulfosuccinimide). In someembodiments, the activating agent is an O-acylisourea-forming agent(e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),dicyclohexylcarbodiimide, and diisopropylcarbodiiimide). In someembodiments, the activating agent is a combination of at least oneacylating agent and at least one O-isourea-forming agents (e.g.,N-hydroxysuccinimide (NHS),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),N-hydroxysulfosuccinimide (sulfo-NHS), and a combination thereof).

In some embodiments, at least one of the first reactive element or thesecond reactive element comprises

In some embodiments, at least one of the first reactive element or thesecond reactive element comprises

wherein X is a halo moiety. For example, X is chloro, bromo, or iodo.

In some embodiments, at least one of the first reactive element or thesecond reactive element comprises

In some embodiments, at least one of the first reactive element or thesecond reactive element comprises

In some embodiments, at least one of the first reactive element or thesecond reactive element comprises

In some embodiments, at least one of the first reactive element or thesecond reactive element is selected from the group consisting of:

wherein

-   R³ is H or C₁-C₆ alkyl; and-   R⁴ is H or trimethylsilyl.

In some embodiments, at least one of the first reactive element or thesecond reactive element comprises

wherein R⁴ is H or trimethylsilyl. In some embodiments, R⁴ is H.

In some embodiments, at least one of the first reactive element or thesecond reactive element is selected from the group consisting of:

wherein R³ is H or C₁-C₆ alkyl. In some embodiments, R³ is H. In someembodiments, R³ is C₁-C₆ alkyl.

In some embodiments, at least one of the first reactive element or thesecond reactive element comprises

wherein R³ is H or C₁-C₆ alkyl. In some embodiments, R³ is H. In someembodiments, R³ is C₁-C₆ alkyl.

In some embodiments, at least one of the first reactive elements or thesecond reactive elements comprises

In some embodiments, at least one of the first reactive elements or thesecond reactive elements comprises

In some embodiments, one of the first reactive elements or the secondreactive elements is selected from the group consisting of:

wherein

-   R¹ is selected from H, C₁-C₆ alkyl, or —SO₃;-   R² is C₁-C₆ alkyl;-   X is a halo moiety;

and the other of the first reactive element or the second reactiveelement is selected from the group consisting of:

wherein

-   R³ is H or C₁-C₆ alkyl; and-   R⁴ is H or trimethylsilyl.

In some embodiments, one of the first reactive elements or the secondreactive elements is selected from the group consisting of

wherein R³ is H or C₁-C₆ alkyl; and the other of the first reactiveelement or the second reactive element is

wherein R⁴ is H or trimethylsilyl. In some embodiments, R³ is H. In someembodiments, R³ is C₁-C₆ alkyl. In some embodiments, R⁴ is H. In someembodiments, R⁴ is trimethylsilyl.

In some embodiments, one of the first reactive element or the secondreactive element is selected from the group consisting of:

wherein

-   R¹ is selected from H, C₁-C₆ alkyl, or —SO₃;-   R² is C₁-C₆ alkyl;-   X is a halo moiety;

and the other of the first reactive element or the second reactiveelement is selected from the group consisting of:

wherein R³ is H or C₁-C₆ alkyl. In some embodiments, R¹ is H. In someembodiments, R¹ is C₁-C₆ alkyl. In some embodiments, R¹ is —SO₃. In someembodiments, R² is methyl. In some embodiments, X is iodo. In someembodiments, R³ is H. In some embodiments, R³ is C₁-C₆ alkyl.

In some embodiments, one of the first reactive elements or the secondreactive elements is selected from the group consisting of:

wherein

-   R¹ is selected from H, C₁-C₆ alkyl, or —SO₃;-   R² is C₁-C₆ alkyl;

and the other of the first reactive elements or the second reactiveelements comprises

wherein R³ is H or C₁-C₆ alkyl. In some embodiments, R¹ is H. In someembodiments, R¹ is C₁-C₆ alkyl. In some embodiments, R¹ is -SO₃. In someembodiments, R² is methyl. In some embodiments, R³ is H. In someembodiments, R³ is C₁-C₆ alkyl.

In some embodiments, one of the first reactive element or the secondreactive element is selected from the group consisting of:

wherein X is a halo moiety; and the other of the first reactive elementor the second reactive element comprises

some embodiments, X is bromo. In some embodiments, X is iodo.

In some embodiments, one of the first reactive element or the secondreactive element is selected from the group consisting of

and the other of the first reactive element or the second reactiveelement comprises

The term “halo” refers to fluoro (F), chloro (Cl), bromo (Br), or iodo(I).

The term “alkyl” refers to a hydrocarbon chain that may be a straightchain or branched chain, containing the indicated number of carbonatoms. For example, C₁₋₁₀ indicates that the group may have from 1 to 10(inclusive) carbon atoms in it. Non-limiting examples include methyl,ethyl, iso-propyl, tert-butyl, n-hexyl.

The term “haloalkyl” refers to an alkyl, in which one or more hydrogenatoms is/are replaced with an independently selected halo.

The term “alkoxy” refers to an —O—alkyl radical (e.g., -OCH₃).

The term “alkylene” refers to a divalent alkyl (e.g., —CH₂—).

The term “alkenyl” refers to a hydrocarbon chain that may be a straightchain or branched chain having one or more carbon-carbon double bonds.The alkenyl moiety contains the indicated number of carbon atoms. Forexample, C₂₋₆ indicates that the group may have from 2 to 6 (inclusive)carbon atoms in it.

The term “alkynyl” refers to a hydrocarbon chain that may be a straightchain or branched chain having one or more carbon-carbon triple bonds.The alkynyl moiety contains the indicated number of carbon atoms. Forexample, C₂₋₆ indicates that the group may have from 2 to 6 (inclusive)carbon atoms in it.

The term “aryl” refers to a 6-20 carbon mono-, bi-, tri- or polycyclicgroup wherein at least one ring in the system is aromatic (e.g.,6-carbon monocyclic, 10-carbon bicyclic, or 14-carbon tricyclic aromaticring system); and wherein 0, 1, 2, 3, or 4 atoms of each ring may besubstituted by a substituent. Examples of aryl groups include phenyl,naphthyl, tetrahydronaphthyl, and the like.

Methods for Non-Covalently Bonding Features to a Substrate Providedherein are methods for the non-covalent bonding of features (e.g.,optically-labeled beads, hydrogel beads, or microsphere beads) to asubstrate.

In some embodiments, features (e.g., beads) are coupled to a substratevia a non-covalent bond between a first affinity group and a secondaffinity group. In some embodiments, the non-covalently-bound features(e.g., beads) substantially form a monolayer of beads (e.g., hydrogelbeads, microsphere beads) on the substrate.

In some embodiments, the features are functionalized with a firstaffinity group, which is directly bound to the features. In someembodiments, the features are functionalized with a first affinitygroup, which is indirectly bound to the beads via a linker. In someembodiments, the linker is a benzophenone. In some embodiments, thelinker is an amino methacrylamide. For example, the linker can be3-aminopropyl methacrylamide. In some embodiments, the linker is a PEGlinker. In some embodiments, the linker is a cleavable linker.

In some embodiments, the substrate is functionalized with a secondaffinity group, which is directly bound to the substrate. In someembodiments, the substrate is functionalized with a second affinitygroup, which is indirectly bound to the beads via a linker. In someembodiments, the linker is a benzophenone. In some embodiments, thelinker is an amino methacrylamide. For example, the linker can be3-aminopropyl methacrylamide. In some embodiments, the linker is a PEGlinker. In some embodiments, the linker is a cleavable linker.

In some embodiments the first affinity group or the second affinitygroup is biotin, and the other of the first affinity group or the secondaffinity group is streptavidin.

In some embodiments, about 99% of the non-covalently-bound beads form amonolayer of beads on the substrate. In some embodiments, about 50% toabout 98% form a monolayer of beads on the substrate. For example, about50% to about 95%, about 50% to about 90%, about 50% to about 85%, about50% to about 80%, about 50% to about 75%, about 50% to about 70%, about50% to about 65%, about 50% to about 60%, or about 50% to about 55% ofthe non-covalently-bound beads form a monolayer of beads on thesubstrate. In some embodiments, about 55% to about 98%, about 60% toabout 98%, about 65% to about 98%, about 70% to about 98%, about 75% toabout 98%, about 80% to about 98%, about 85% to about 98%, about 90% toabout 95%, or about 95% to about 98% of the non-covalently-bound beadsform a monolayer of beads on the substrate. In some embodiments, about55% to about 95%, about 60% to about 90%, about 65% to about 95%, about70% to about 95%, about 75% to about 90%, about 75% to about 95%, about80% to about 90%, about 80% to about 95%, about 85% to about 90%, orabout 85% to about 95% of the non-covalently-bound beads for a monolayerof beads on the substrate.

In some embodiments, the monolayer of beads is a formed in a predefinedarea on the substrate. In some embodiments, the predefined area ispartitioned with physical barriers. For example, divots or wells in thesubstrate. In some embodiments, the predefined area is partitioned usinga photomask. For example, the substrate is coated with a photo-activatedsolution, dried, and then irradiated under a photomask. In someembodiments, the photo-activated solution is UV-activated.

As used herein, an “adhesive” generally refers to a substance used forsticking objects or materials together. Adhesives include, for example,glues, pastes, liquid tapes, epoxy, bioadhesives, gels, and mucilage. Insome embodiments, an adhesive is liquid tape. In some embodiments, theadhesive is glue.

In some embodiments, beads are adhered to a substrate using an adhesive(e.g., liquid tape, glue, paste). In some embodiments, the adhered beadssubstantially form a monolayer of beads on the substrate (e.g., a glassslide). In some embodiments, the beads are hydrogel beads. In someembodiments, the beads are microsphere beads. In some embodiments, thebeads are coated with the adhesive, and then the beads are contactedwith the substrate. In some embodiments, the substrate is coated withthe adhesive, and then the substrate is contacted with the beads. Insome embodiments, both the substrate is coated with the adhesive and thebeads are coated with the adhesive, and then the beads and substrate arecontacted with one another.

In some embodiments, about 99% of the adhered beads form a monolayer ofbeads on the substrate. In some embodiments, about 50% to about 98% forma monolayer of beads on the substrate. For example, about 50% to about95%, about 50% to about 90%, about 50% to about 85%, about 50% to about80%, about 50% to about 75%, about 50% to about 70%, about 50% to about65%, about 50% to about 60%, or about 50% to about 55% of the adheredbeads form a monolayer of beads on the substrate. In some embodiments,about 55% to about 98%, about 60% to about 98%, about 65% to about 98%,about 70% to about 98%, about 75% to about 98%, about 80% to about 98%,about 85% to about 98%, about 90% to about 95%, or about 95% to about98% of the adhered beads form a monolayer of beads on the substrate. Insome embodiments, about 55% to about 95%, about 60% to about 90%, about65% to about 95%, about 70% to about 95%, about 75% to about 90%, about75% to about 95%, about 80% to about 90%, about 80% to about 95%, about85% to about 90%, or about 85% to about 95% of the adhered beads for amonolayer of beads on the substrate.

In some embodiments, beads can be deposited onto a biological samplesuch that the deposited beads form a monolayer of beads on thebiological sample (e.g., over or under the biological sample). In someembodiments, beads deposited on the substrate can self-assemble into amonolayer of beads that saturate the intended surface area of thebiological sample under investigation. In this approach, bead arrays canbe designed, formulated, and prepared to evaluate a plurality ofanalytes from a biological sample of any size or dimension. In someembodiments, the concentration or density of beads (e.g., gel beads)applied to the biological sample is such that the area as a whole, orone or more regions of interest in the biological sample, is saturatedwith a monolayer of beads. In some embodiments, the beads are contactedwith the biological sample by pouring, pipetting, spraying, and thelike, onto the biological sample. Any suitable form of bead depositioncan be used.

In some embodiments, the biological sample can be confined to a specificregion or area of the array. For example, a biological sample can beaffixed to a glass slide and a chamber, gasket, or cage positioned overthe biological sample to act as a containment region or frame withinwhich the beads are deposited. As will be apparent, the density orconcentration of beads needed to saturate an area or biological samplecan be readily determined by one of ordinary skill in the art (e.g.,through microscopic visualization of the beads on the biologicalsample). In some embodiments, the bead array contains microfluidicchannels to direct reagents to the spots or beads of the array.

Feature Geometric Attributes

Features on an array can have a variety of sizes. In some embodiments, afeature of an array can have an average diameter or maximum dimensionbetween 500 nm µm to 100 µm. For example, between 500 nm to 2 µm, 1 µmto 3 µm, 1 µm to 5 µm, 1 µm to 10 µm, 1 µm to 20 µm, 1 µm to 30 µm, 1 µmto 40 µm, 1 µm to 50 µm, 1 µm to 60 µm, 1 µm to 70 µm, 1 µm to 80 µm, 1µm to 90 µm, 90 µm to 100 µm, 80 µm to 100 µm, 70 µm to 100 µm, 60 µm to100 µm, 50 µm to 100 µm, 40 µm to 100 µm, 30 µm to 100 µm, 20 µm to 100µm, 10 µm to 100 µm, about 40 µm to about 70 µm, or about 50 µm to about60 µm. In some embodiments, the feature has an average diameter ormaximum dimension between 30 µm to 100 µm, 40 µm to 90 µm, 50 µm to 80µm, 60 µm to 70 µm, or any range within the disclosed sub-ranges. Insome embodiments, the feature has an average diameter or maximumdimension no larger than 95 µm, 90 µm, 85 µm, 80 µm, 75 µm, 70 µm, 65µm, 60 µm, 55 µm, 50 µm, 45 µm, 40 µm, 35 µm, 30 µm, 25 µm, 20 µm, 15µm, 14 µm, 13 µm, 12 µm, 11 µm, 10 µm, 9 µm, 8 µm, 7 µm, 6 µm, 5 µm, 4µm, 3 µm, 2 µm, or 1 µm. In some embodiments, the feature has an averagediameter or maximum dimension of approximately 65 µm. In someembodiments, the feature has an average diameter or maximum distance ofapproximately 55 µm.

In some embodiments, the size and/or shape of a plurality of features ofan array are approximately uniform. In some embodiments, the size and/orshape of a plurality of features of an array is not uniform. Forexample, in some embodiments, features in an array can have an averagecross-sectional dimension, and a distribution of cross-sectionaldimensions among the features can have a full-width and half-maximumvalue of 0% or more (e.g., 5% or more, 10% or more, 20% or more, 30% ormore, 40% or more, 50% or more, 70% or more, or 100% or more) of theaverage cross-sectional dimension for the distribution.

In certain embodiments, features in an array can have an averagecross-sectional dimension of between about 1 µm and about 10 µm. Thisrange in average feature cross-sectional dimension corresponds to theapproximate diameter of a single mammalian cell. Thus, an array of suchfeatures can be used to detect analytes at, or below, mammaliansingle-cell resolution.

In some embodiments, a plurality of features has a mean diameter or meanmaximum dimension of about 0.1 µm to about 100 µm (e.g., about 0.1 µm toabout 5 µm, about 1 µm to about 10 µm, about 1 µm to about 20 µm, about1 µm to about 30 µm, about 1 µm to about 40 µm, about 1 µm to about 50µm, about 1 µm to about 60 µm, about 1 µm to about 70 µm, about 1 µm toabout 80 µm, about 1 µm to about 90 µm, about 90 µm to about 100 µm,about 80 µm to about 100 µm, about 70 µm to about 100 µm, about 60 µm toabout 100 µm, about 50 µm to about 100 µm, about 40 µm to about 100 µm,about 30 µm to about 100 µm, about 20 µm to about 100 µm, or about 10 µmto about 100 µm). In some embodiments, the plurality of features has amean diameter or mean maximum dimension between 30 µm to 100 µm, 40 µmto 90 µm, 50 µm to 80 µm, 60 µm to 70 µm, or any range within thedisclosed sub-ranges. In some embodiments, the plurality of features hasa mean diameter or a mean maximum dimension no larger than 95 µm, 90 µm,85 µm, 80 µm, 75 µm, 70 µm, 65 µm, 60 µm, 55 µm, 50 µm, 45 µm, 40 µm, 35µm, 30 µm, 25 µm, 20 µm, 15 µm, 14 µm, 13 µm, 12 µm, 11 µm, 10 µm, 9 µm,8 µm, 7 µm, 6 µm, 5 µm, 4 µm, 3 µm, 2 µm, or 1 µm. In some embodiments,the plurality of features has a mean average diameter or a mean maximumdimension of approximately 65 µm, approximately 60 µm, approximately 55µm, approximately 50 µm, approximately 45 µm, approximately 40 µm,approximately 35 µm, approximately 30 µm, approximately 25 µm,approximately 20 µm, approximately 15 µm, approximately 10 µm,approximately 5 µm, approximately 4 µm, approximately 3 µm,approximately 2 µm, or approximately 1 µm.

(IV) Array Geometric Attributes

In some embodiments, an array includes a plurality of features. Forexample, an array includes between 4,000 and 50,000 features, or anyrange within 4,000 to 40,000 features. For example, an array includesbetween 4,000 to 35,000 features, 4,000 to 30,000 features, 4,000 to25,000 features, 4,000 to 20,000 features, 4,000 to 15,000 features,4,000 to 10,000 features, 4,000 to 6,000 features, or 4,400 to 6,000features. In some embodiments, the array includes between 4,100 and5,900 features, between 4,200 and 5,800 features, between 4,300 and5,700 features, between 4,400 and 5,600 features, between 4,500 and5,500 features, between 4,600 and 5,400 features, between 4,700 and5,300 features, between 4,800 and 5,200 features, between 4,900 and5,100 features, or any range within the disclosed sub-ranges. Forexample, the array can include about 4,000 features, about 4,200features, about 4,400 features, about 4,800 features, about 5,000features, about 5,200 features, about 5,400 features, about 5,600features, or about 6,000 features, about 10,000 features, about 20,000features, about 30,000 features, about 40,000 features, or about 50,000features. In some embodiments, the array comprises at least 4,000features. In some embodiments, the array includes approximately 5,000features.

In some embodiments, features within an array have an irregulararrangement or relationship to one another, such that no discernablepattern or regularity is evident in the geometrical spacingrelationships among the features. For example, features within an arraymay be positioned randomly with respect to one another. Alternatively,features within an array may be positioned irregularly, but the spacingsmay be selected deterministically to ensure that the resultingarrangement of features is irregular.

In some embodiments, features within an array are positioned regularlywith respect to one another to form a pattern. A wide variety ofdifferent patterns of features can be implemented in arrays. Examples ofsuch patterns include, but are not limited to, square arrays offeatures, rectangular arrays of features, hexagonal arrays of features(including hexagonal close-packed arrays), radial arrays of features,spiral arrays of features, triangular arrays of features, and moregenerally, any array in which adjacent features in the array are reachedfrom one another by regular increments in linear and/or angularcoordinate dimensions.

In some embodiments, features within an array are positioned with adegree of regularity with respect to one another such that the array offeatures is neither perfectly regular nor perfectly irregular (i.e., thearray is “partially regular”). For example, in some embodiments,adjacent features in an array can be separated by a displacement in oneor more linear and/or angular coordinate dimensions that is 10% or more(e.g., 20% or more, 30% or more, 40% or more, 50% or more, 60% or more,70% or more, 80% or more, 90% or more, 100% or more, 110% or more, 120%or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% ormore, 180% or more, 190% or more, 200% or more) of an averagedisplacement or a nominal displacement between adjacent features in thearray. In certain embodiments, the distribution of displacements (linearand/or angular) between adjacent features in an array has a full-widthat half-maximum of between 0% and 200% (e.g., between 0% and 100%,between 0% and 75%, between 0% and 50%, between 0% and 25%, between 0%and 15%, between 0% and 10%) of an average displacement or nominaldisplacement between adjacent features in the array.

In some embodiments, arrays of features can have a variable geometry.For example, a first subset of features in an array can be arrangedaccording to a first geometrical pattern, and a second subset offeatures in the array can be arranged according to a second geometricalpattern that is different from the first pattern. Any of the patternsdescribed above can correspond to the first and/or second geometricalpatterns, for example.

In general, arrays of different feature densities can be prepared byadjusting the spacing between adjacent features in the array. In someembodiments, the geometric center-to-center (e.g., pitch) spacingbetween adjacent features in an array is between 100 nm to 10 µm, 500 nmto 2 µm, 1 µm to 5 µm, and 20 µm to 200 µm. For example, thecenter-to-center spacing can be between 100 nm to 10 µm, 500 nm to 2 µm,1 µm, to 5 µm, 20 µm to 40 µm, 20 µm to 60 µm, 20 µm to 80 µm, 80 µm to100 µm, 100 µm to 120 µm, 120 µm to 140 µm, 140 µm to 160 µm, 160 µm to180 µm, 180 µm to 200 µm, 60 µm to 100 µm, or 40 µm to 100 µm, 50 µm to150 µm, 80 µm to 120 µm, or 90 µm to 110 µm. In some embodiments, thepitch between adjacent array features is between 30 µm and 100 µm, 40 µmand 90 µm, 50 µm and 80 µm, 60 µm and 70 µm, 80 µm and 120 µm, or anyrange within the disclosed sub-ranges. In some embodiments, the pitchbetween adjacent array features of an array is approximately 65 µm,approximately 60 µm, approximately 55 µm, approximately 50 µm,approximately 45 µm, approximately 40 µm, approximately 35 µm,approximately 30 µm, approximately 25 µm, approximately 20 µm,approximately 15 µm, approximately 10 µm, approximately 5 µm,approximately 4 µm, approximately 3 µm, approximately 2 µm, orapproximately 1 µm. In some embodiments, the pitch between adjacentarray features of an array is less than 100 µm.

An array of features can have any appropriate resolution. In someembodiments, an array of features can have a spatially constant (e.g.,within a margin of error) resolution. In general, an array with aspatially consistent resolution is an array in which the pitch betweenadjacent features in the array is constant (e.g., within a margin oferror). Such arrays can be useful in a variety of applications. In someembodiments, an array of features can have a spatially varyingresolution. In general, an array with a spatially varying resolution isan array in which the center-to-center spacing (e.g., pitch) (alonglinear, angular, or both linear and angular coordinate dimensions)between adjacent features in the array varies. Such arrays can be usefulin a variety of applications. For example, in some embodiments,depending upon the spatial resolution at which the sample is to beinvestigated, the sample can be selectively associated with the portionof the array that corresponds approximately to the desired spatialresolution of the measurement.

In some embodiments, it may be useful to describe the resolution of anarray of features by functional aspects, for example, the number ofreads that can be carried out per feature (which can be a proxy forsequencing saturation), the number of transcripts that can be detectedper feature, or the number of genes that can be detected per feature.For example, in some embodiments, the number of reads that can beperformed per feature is between 50,000 and 1,000,000. For example, thenumber of reads that can be performed per feature can be between 50,000and 100,000, 50,000 and 150,000, 50,000 and 200,000, 50,000 and 250,000,50,000 and 300,000, 50,000 and 350,000, 50,000 and 400,000, 50,000 and500,000, 50,000 and 550,000, 50,000 and 600,000, 50,000 and 650,000,50,000 and 700,000, 50,000 and 750,000, 50,000 and 800,000, 50,000 and850,000, 50,000 and 900,000, 50,000 and 950,000, 50,000 and 1,000,000,100,000 to 500,000, 150,000 to 500,000, 200,000 to 500,000, 250,000 to500,000, 300,000 and 500,000, 350,000 and 500,000, 400,000 and 500,000,450,000 and 500,000, 150,000 to 250,000, or 300,000 to 400,000. In someembodiments, the number of reads that can be performed per feature isabout 70,000. In some embodiments, the number of reads that can beperformed per feature is about 170,000. In some embodiments, the numberreads that can be performed per feature is about 330,000. In someembodiments, the number reads that can be performed per feature is about500,000. In some embodiments, the number reads that can be performed perfeature is about 800,000.

In some embodiments, the number of transcripts that can be detected perfeature is between 20,000 and 200,000. For example, in some embodiments,the number of transcripts that can be detected per feature can bebetween 20,000 and 30,000, 20,000 and 40,000, 20,000 and 50,000, 30,000and 60,000, 40,000 and 60,000, 50,000 and 60,000, 20,000 and 100,000,30,000 and 100,000, 40,000 and 200,000, 50,000 and 200,000, or 30,000and 200,000. In some embodiments, the number of transcripts that can bedetected per feature is about 40,000. In some embodiments, the number oftranscripts that can be detected per feature is about 60,000. In someembodiments, the number of transcripts that can be detected per featureis about 80,000. In some embodiments, the number of transcripts that canbe detected per feature is about 100,000.

In some embodiments, the number of genes that can be detected perfeature is between 1,000 and 5,000. For example, the number of genesthat can be detected per feature can be between 1,000 and 1,500, 1,000and 2,000, 1,000 and 2,500, 1,000 and 3,000, 1,000 and 3,500, 1,000 and4,000, 1,000 and 4,500, 1,500 and 5,000, 2,000 and 5,000, 2,500 and5,000, 3,000 and 5,000, 3,500 and 5,000, 4,000 and 5,000, 4,500 and5,000, 1,500 and 2,500, 2,500 and 3,500, or 3,500 and 4,000. In someembodiments, the number of genes that can be detected per feature isabout 2,000. In some embodiments, the number of genes that can bedetected per feature is about 3,000. In some embodiments, the number ofgenes that can be detected per feature is about 4,000.

In some embodiments, it may be useful to describe the resolution of anarray of features by functional aspects, for example, the number of UMIcounts per feature. For example, in some embodiments, the number of UMIcounts that can be performed per feature is between 1,000 and 50,000. Insome embodiments, the number of UMI counts can be averaged to obtain amean UMI per feature. In some embodiments, the number of UMI counts canbe averaged to obtain a median UMI count per feature. For example, themedian UMI count per feature can be between 1,000 and 50,000, 1,000 and40,000, 1,000 and 30,000, 1,000 and 20,000, 1,000 and 10,000, 1,000 and5,000. In some embodiments, the median UMI count per feature is about5,000. In some embodiments, the median UMI count per feature is about10,000.

These components can be used to determine the sequencing saturation ofthe array. The sequencing saturation can be a measure of the librarycomplexity and sequencing depth. For example, different cell types willhave different amounts of RNA, thus different number of transcripts,influencing library complexity. Additionally, sequencing depth isrelated to the number of sequencing reads. In some embodiments, theinverse of sequencing saturation is the number of additional reads itwould take to detect a new transcript. One way of measuring thesequencing saturation of an array is to determine the number of reads todetect a new UMI. For example, if a new UMI is detected every 2 reads ofthe feature, the sequencing saturation would be 50%. As another example,if a new UMI is detected every 10 reads of a feature, the sequencingsaturation would be 90%.

Arrays of spatially varying resolution can be implemented in a varietyof ways. In some embodiments, for example, the pitch between adjacentfeatures in the array varies continuously along one or more linearand/or angular coordinate directions. Thus, for a rectangular array, thespacing between successive rows of features, between successive columnsof features, or between both successive rows and successive columns offeatures, can vary continuously.

In certain embodiments, arrays of spatially varying resolution caninclude discrete domains with populations of features. Within eachdomain, adjacent features can have a regular pitch. Thus, for example,an array can include a first domain within which adjacent features arespaced from one another along linear and/or angular coordinatedimensions by a first set of uniform coordinate displacements, and asecond domain within which adjacent features are spaced from one anotheralong linear and/or angular coordinate dimensions by a second set ofuniform coordinate displacements. The first and second sets ofdisplacements differ in at least one coordinate displacement, such thatadjacent features in the two domains are spaced differently, and theresolution of the array in the first domain is therefore different fromthe resolution of the array in the second domain.

In some embodiments, the pitch of array features can be sufficientlysmall such that array features are effectively positioned continuouslyor nearly continuously along one or more array dimensions, with littleor no displacement between array features along those dimensions. Forexample, in a feature array where the features correspond to regions ofa substrate (i.e., oligonucleotides are directly bound to thesubstrate), the displacement between adjacent oligonucleotides can bevery small - effectively, the molecular width of a singleoligonucleotide. In such embodiments, each oligonucleotide can include adistinct spatial barcode such that the spatial location of eacholigonucleotide in the array can be determined during sample analysis.Arrays of this type can have very high spatial resolution, but may onlyinclude a single oligonucleotide corresponding to each distinct spatiallocation in a sample.

In general, the size of the array (which corresponds to the maximumdimension of the smallest boundary that encloses all features in thearray along one coordinate direction) can be selected as desired, basedon criteria such as the size of the sample, the feature diameter, andthe density of capture probes within each feature. For example, in someembodiments, the array can be a rectangular or square array for whichthe maximum array dimension along each coordinate direction is 10 mm orless (e.g., 9 mm or less, 8 mm or less, 7 mm or less, 6 mm or less, 5 mmor less, 4 mm or less, 3 mm or less). Thus, for example, a square arrayof features can have dimensions of 8 mm by 8 mm, 7 mm by 7 mm, 5 mm by 5mm, or be smaller than 5 mm by 5 mm.

(V) Bead Arrays

As used herein, the term “bead array” refers to an array that includes aplurality of beads as the features in the array. In some embodiments,two or more beads are dispersed onto a substrate to create an array,where each bead is a feature on the array. In some embodiments, thebeads are attached to a substrate. For example, the beads can optionallyattach to a substrate such as a microscope slide and in proximity to abiological sample (e.g., a tissue section that includes cells). Thebeads can also be suspended in a solution and deposited on a surface(e.g., a membrane, a tissue section, or a substrate (e.g., a microscopeslide)). Beads can optionally be dispersed into wells on a substrate,e.g., such that only a single bead is accommodated per well.

Examples of arrays of beads on or within a substrate include beadslocated in wells such as the BeadChip array (available from IlluminaInc., San Diego, CA), arrays used in sequencing platforms from 454LifeSciences (a subsidiary of Roche, Basel, Switzerland), and array usedin sequencing platforms from Ion Torrent (a subsidiary of LifeTechnologies, Carlsbad, CA). Examples of bead arrays are described in,e.g., U.S. Pats. Nos. 6,266,459; 6,355,431; 6,770,441; 6,859,570;6,210,891; 6,258,568; and 6,274,320; U.S. Pat. Application PublicationNos. 2009/0026082; 2009/0127589; 2010/0137143; 2019/0177777; and2010/0282617; and PCT Patent Application Publication Nos. WO 00/063437and WO 2016/162309, the entire contents of each of which is incorporatedherein by reference.

In some embodiments, the bead array includes a plurality of beads. Forexample, the bead array can include at least 10,000 beads (e.g., atleast 100,000 beads, at least 1,000,000 beads, at least 5,000,000 beads,at least 10,000,000 beads). In some embodiments, the plurality of beadsincludes a single type of bead (e.g., substantially uniform in volume,shape, and other physical properties, such as translucence). In someembodiments, the plurality of beads includes two or more types ofdifferent beads.

Bead arrays can be generated by attaching beads (e.g., barcoded beads)to a substrate in a regular pattern, or an irregular arrangement. Insome embodiments, the barcode sequences are known before attaching themto the substrate. In some embodiments, the barcode sequences are notknown before attaching them to the substrate. Beads can be attached toselective regions on a substrate by, e.g., selectively activatingregions on the substrate to allow for attachment of the beads.Activating selective regions on the substrate can include activating ordegrading a coating (e.g., a conditionally removable coating asdescribed herein) at the selective regions where the coating has beenapplied on the substrate, rendering the selective regions morepermissive to bead attachment as compared to regions outside of theselected regions. The regions that are rendered more permissive for beadattachment can be configured to fit only one bead or multiple beads(e.g., limited by well size or surface patterning, such as fabricationtechniques). Beads bound to the selected regions can form atwo-dimensional array on the substrate. The substrate can be uniformlyor non-uniformly coated with the coating. The beads can be any suitablebeads described herein, including beads that are attached to one or morespatial barcodes. Beads can be attached to the selected regionsaccording to any of the methods suitable for attaching beads tosubstrates described herein, such as through covalent bonds,non-covalent bonds, or chemical linkers.

Any variety of suitable patterning techniques can be used to attachbeads to a substrate surface. In some embodiments, in a non-limitingway, physical techniques such as inkjet printing, optical andoptoelectronic cell trapping, laser-based patterning, acousticpatterning, dielectrophoresis, or magnetic techniques can be used topattern the substrate. Alternatively, chemical and/or physiochemicaltechniques can be used such as, in a non-limiting way, surface chemistrymethods, microcontact printing, microwells and filtration, DUVpatterning, or patterning in microfluidic devices combined withmicrocontact printing (See, e.g., Martinez-Rivas, A., Methods ofmicropatterning and manipulation of cells for biomedical applications,Micromachines (Basel) 8, (2017), which in is incorporated herein byreference).

The coating can be photoreactive, and selectively activating ordegrading the coating involves exposing selected regions of the coatingto light or radiation. Selectivity can be achieved through theapplication of photomasks. Regions of the coating that are exposed tolight can be rendered more permissive for bead attachment (e.g., moreadhesive), as compared to regions not exposed to light (e.g., regionsprotected from the light by a photomask). When applied to the substrate,the beads thus preferentially attach to the more permissive regions onthe substrate, and un-attached beads can optionally be removed from thesubstrate. The light source and/or the photomask can be adjusted toallow further sites on the substrate to become more permissive for beadattachment, allowing additional beads to be attached at these sites.Alternatively, a different light source, or a different photomask can beapplied. The process of photopatterning thus allows beads to be attachedat pre-determined locations on the substrate, thereby generating a beadarray.

Beads can be attached iteratively, e.g., a subset of the beads can beattached at one time, and the process can be repeated to attach one ormore additional subsets of beads. In some embodiments, the size of theactivated spot (e.g., spot on the substrate) is smaller than the size ofa bead. For example, a bead can be attached to the activated substrate(e.g., spot) such that only a single bead attaches to the activatedsubstrate. In some embodiments, the substrate can be washed to removeunbound beads. In some embodiments, the substrate can be activated in asecond location and a second bead can be attached to the activatedsubstrate surface. This process can be done iteratively to attach beadsto the entire substrate, or a portion thereof. Alternatively, beads canbe attached to the substrate all in one step. Furthermore, methods ofattaching beads to a substrate are known in the art. Any suitable methodcan be used, including, in a non-limiting way, specific chemical bonds,non-specific chemical bonds, linkers, physically trapping the beads(e.g., polymer, hydrogel), or any of the methods described herein.

An exemplary workflow for generating a bead array can includeselectively rendering a first set of one or more selected regions on acoated substrate to be more permissive for bead attachment as comparedto regions outside of the selected regions, applying a plurality ofbeads to the array and allowing the beads to attach to the first set ofselected regions, optionally removing un-attached beads, rendering asecond set of one or more selected regions more permissive to beadattachment as compared to regions outside the second set of selectedregions, applying a plurality of beads and allowing the beads to attachto the second set of selected regions, and optionally removing theun-attached beads. This iterative process can be carried out for anynumber of times to generate a patterned bead array.

Another exemplary process includes activating a first region on a coatedsubstrate and exposing the activated first region to a plurality ofbarcoded beads, so that a first set of one or more beads are bound tothe first region; and activating a second region on the coated substrateand exposing the activated second region to a plurality of barcodedbeads, so that a second set of one or more beads are bound to the secondregion, wherein the first set of one or more beads comprise an identicalfirst oligonucleotide sequence unique to the first region of the surfaceof the substrate, and the second set of one or more beads comprise anidentical second oligonucleotide sequence unique to the second region ofthe surface of the substrate, and wherein the first and secondoligonucleotide sequences are different. Additional regions on thecoated substrate may be activated and exposed to additional barcodedbeads. Each set of barcoded beads can include an oligonucleotidesequence that is different from all other sets of barcoded beads andthat is unique to the location of the activated region. Additionally,the first set of one or more beads and the second set of one or morebeads can be different. In other words, the first set of one or morebeads and the second set of one or more beads can have different surfacechemistries, different compositions (e.g., solid bead, gel bead, silicabead)(e.g., nanoparticles vs microparticles), and/or physical volumes.In some embodiments, a third set of one or more beads, a fourth set ofone or more beads, a fifth set of one or more beads or more can havedifferent surface chemistries, different compositions (e.g., solid bead,gel bead, silica bead)(e.g., nanoparticles vs microparticles), and/orphysical volumes can be attached to the substrate surface. The methodsmay include removing the beads that do not bind to the first, second,and/or any of the additional regions. In some embodiments, removing thebeads comprise washing the beads off the surface of the substrate. Theremoving may be carried out after each round of or after several roundsof activating a region (e.g., first, second or additional regions on thesurface of the substrate), and binding of beads to the activated region.In some instances, each bead is bound to the substrate at a singlelocation. The beads bound to the first, second, and additional regionscan form a two-dimensional array of beads on the substrate.

A photoreactive coating can comprise a plurality of photoreactivemolecules, which can undergo a chemical reaction (e.g., hydrolysis,oxidation, photolysis) when exposed to light of certain wavelengths orrange of wavelengths. A photo-reactive molecule can become reactive whenexposed to light and can react with other molecules and form chemicalbonds with other molecules.

The coating can comprise a polymer, and activating selected regions onthe substrate include modifying the polymer at the respective regions.Modifying the polymer includes, for example, photochemically modifyingthe polymer by exposing the polymer to radiation or light. Alternativelyor additionally, modifying the polymer can include chemically modifyingthe polymer by contacting the polymer with one or more chemicalreagents. In some instances, the coating is a hydrogel. In someinstances, the coating comprises a photoreactive polymer. Exemplaryphoto-reactive polymers include poly(ethylene glycol) (PEG)-basedpolymers, poly(L-lysine) (PLL)-based polymer, copolymer comprisingfunctionalized or unfunctionalized units of PEG and PLL (e.g.,poly-L-lysine-grafted-polyethylene glycol (PLL-g-PEG)), andmethacrylated gelatin (GelMA) polymers.

Beads can also be attached to selective regions on a substrate byselectively crosslinking beads to a coating that has been applied on thesubstrate. For example, a plurality of beads can be applied to asubstrate having a photocrosslinkable coating, and upon crosslinking ofa subset of the beads to the coating, the non-cross-linked beads can beremoved, leaving only the cross-linked beads on the substrate. Theprocess can be repeated multiple times. The coating can include aphoto-crosslinkable polymer. Exemplary photo-crosslinkable polymers aredescribed, e.g., in Shirai, Polymer Journal 46:859-865 (2014), Ravve,Photocrosslinkable Polymers, Light-Associated Reactions of SyntheticPolymers. Springer, New York, NY (2006), and Ferreira et al.Photocrosslinkable Polymers for Biomedical Applications, BiomedicalEngineering - Frontiers and Challenges, Prof. Reza Fazel (Ed.), ISBN:978-953-307-309-5 (2011), each of which are herein incorporated byreference in its entirety.

Suitable light sources for activating, degrading or crosslinking thecoating as described herein include, but are not limited to, Ultraviolet(UV) light (e.g., 250-350 nm or 350-460 nm UV light) and visible light(e.g., broad spectrum visible light). A Digital Micromirror Device (DMD)can also be used to provide the light source.

The distance between a first pair of adjacent selected regions accordingto the methods described herein can be the same or different from asecond pair of adjacent selected regions.

Barcoded beads, or beads comprising a plurality of barcoded probes, canbe generated by first preparing a plurality of barcoded probes on asubstrate, depositing a plurality of beads on the substrate, andgenerating probes attached to the beads using the probes on thesubstrate as a template.

Large scale commercial manufacturing methods allow for millions ofoligonucleotides to be attached to an array. Commercially availablearrays include those from Roche NimbleGen, Inc., (Wisconsin) andAffymetrix (ThermoFisher Scientific).

In some embodiments, arrays can be prepared according to the methods setforth in WO 2012/140224, WO 2014/060483, WO 2016/162309, WO 2017/019456,WO 2018/091676, and WO 2012/140224, and U.S. Pat. Application No.2018/0245142. The entire contents of each of the foregoing documents areherein incorporated by reference.

In some embodiments, a bead array is formed when beads are embedded in ahydrogel layer where the hydrogel polymerizes and secures the relativebead positions. The bead-arrays can be pre-equilibrated and combinedwith reaction buffers and enzymes (e.g., reverse-transcription mix). Insome embodiments, the bead arrays can be stored (e.g., frozen) long-term(e.g., days) prior to use.

(VI) Flexible Arrays

A “flexible array” includes a plurality of spatially-barcoded featuresattached to, or embedded in, a flexible substrate (e.g., a membrane, ahydrogel, or tape) placed onto, or proximal to, a biological sample. Insome embodiments, a flexible array includes a plurality ofspatially-barcoded features embedded within a hydrogel.

Flexible arrays can be highly modular. In some embodiments,spatially-barcoded features (e.g., beads) can be loaded onto a substrate(e.g., a slide) to produce a high-density self-assembled array. In someembodiments, the features (e.g., beads) can be loaded onto the substratewith a flow cell. In some embodiments, the features (e.g., beads) areembedded in a hydrogel (e.g., a hydrogel pad or layer placed on top ofthe self-assembled features). In some embodiments, the hydrogel canpolymerize, thereby securing the features in the hydrogel. In someembodiments, the hydrogel containing the features can be removed fromthe substrate and used as a flexible array. In some embodiments, theflexible array can be deconvolved by optical sequencing or any othermethod described herein. In some embodiments, the features (e.g., beads)can be about 1 µm to about 25 µm in diameter. In some embodiments, about25 µm diameter features in the flexible array can provide forapproximately 1000 DPI and about 1 megapixel resolution. In someembodiments, the features (e.g., beads) can be about 13.2 µm indiameter. In some embodiments, the about 13.2 µm beads in the flexiblearray can provide for approximately 1920x1080 resolution.

Flexible arrays generated according to any of the methods describedherein (e.g., beads embedded within a hydrogel) can contain athermolabile polymer. In some embodiments, flexible arrays havingthermolabile beads can be contacted with a biological sample. In someembodiments, a region of interest in the biological sample can beidentified such that an infrared laser can be used to select a region ofinterest. In some embodiments, the infrared laser can cause the flexiblearray (e.g., thermolabile beads) to deform and become adhesive. In someembodiments, the adhesive portion of the flexible array can adhere(e.g., bind) to the region of interest (e.g., cells) directly above orunderneath. The process of identifying a region of interest, applying aninfrared laser to the region of interest, and adhering the underlyingbiological sample (e.g., cells) to the flexible array can iterativelyrepeated. In some embodiments, the flexible array can be removed suchthat only the adhered biological sample (e.g., cells) from the one ormore regions of interest can also be removed with the flexible array. Insome embodiments, the flexible array and the adhered biological samplecan be further processed (e.g., amplified, quantitated, and/orsequenced) according to any method described herein.

Flexible arrays can be pre-equilibrated with reaction buffers andenzymes at functional concentrations (e.g., a reverse-transcriptionmix). In some embodiments, the flexible arrays can be stored forextended periods (e.g., days) or frozen until ready for use. In someembodiments, permeabilization of biological samples (e.g., a tissuesection) can be performed with the addition of enzymes/detergents priorto contact with the flexible array. In some embodiments, the flexiblearray can be placed directly on the sample, or placed in indirectcontact with the sample (e.g., with an intervening layer or substancebetween the biological sample and the flexible bead-array). In someembodiments, the flexible array can be mechanically applied (e.g.,pressed downward or compressed between two surfaces) on the biologicalsample to enhance analyte capture. In some embodiments, a flexible arraycan be applied to the side of a biological sample. For example, abiological sample can be cut (e.g., sliced) in any direction and aflexible array can be applied to the exposed analytes. In someembodiments, the flexible array can be dissolvable (e.g., via heat,chemical, or enzymatic disruption). In some embodiments, once a flexiblearray is applied to the sample, reverse transcription and targetedcapture of analytes can be performed on microspheres, or beads of afirst volume and beads of a second volume, or any of the beads describedherein. In some embodiments once a flexible array is applied to thebiological sample and allowed to capture analytes, the flexible arraycan be removed (e.g., peeled) from the biological sample for furtherprocessing (e.g., amplification, quantitation, and/or sequencing)according to any method described herein.

Flexible arrays can also be used with any of the methods (e.g., activecapture methods such as electrophoresis) described herein. For example,flexible arrays can be contacted with a biological sample on aconductive substrate (e.g., an indium tin oxide coated glass slide),such that an electric field can be applied to the conductive substrateto facilitate migration of analytes through, across, within, or in thedirection of the flexible array. Additionally and alternatively,flexible arrays can be contacted to a biological sample in anelectrophoretic assembly (e.g., electrophoretic chamber), such that anelectric field can be applied to migrate analytes in the direction ofthe flexible array or across, through, or within the flexible array.

In some embodiments, a flexible array can be generated with theassistance of a substrate holder (e.g., any array alignment device). Forexample, a spatially-barcoded bead array can be placed in oneplaceholder of the substrate holder and second substrate (e.g., a glassslide) can be placed in the second placeholder of the substrate holder.In some embodiments, the array is optionally optically decoded and a gelprepolymer solution is introduced between the spatially-barcoded beadarray and second substrate. In some embodiments, the substrate holder isclosed such that the second substrate is on top (e.g., above, parallelto) the spatially-barcoded bead array. The gel prepolymer solution canbe polymerized by any method described herein and result inspatially-barcoded features cross-linked in the hydrogel, therebygenerating a flexible array. In some embodiments, the substrate holdercan be opened and the second substrate with the hydrogel and thespatially-barcoded cross-linked features can be removed from thesubstrate holder (the flexible array optionally can be removed from thesecond substrate) for use in spatial analysis by any of the methodsdescribed herein.

(VII) Shrinking Hydrogel Features / Arrays

As used herein “shrinking” or “reducing the size” of a hydrogel refersto any process causing the hydrogel to physically contract and/or thesize of the hydrogel to decrease in volume. For example, the scaffold ofthe gel may shrink or “implode” upon solvent removal (see, e.g., Longand Williams. Science. 2018; 362(6420):1244-1245, and Oran et al.Science 2018; 362(6420): 1281-1285; each of which is incorporated hereinby reference in its entirety). As another example, the process to shrinkor reduce the volume of a hydrogel may be one that removes water (i.e.,a dehydrating process) from the hydrogel. There are many methods knownto one of skill in the art for shrinking or reducing the volume of ahydrogel. Non-limiting examples of a method to shrink or reduce thevolume of a hydrogel include exposing the hydrogel to one or more of: adehydrating solvent, a salt, heat, a vacuum, lyophilization,desiccation, filtration, air-drying, or combinations thereof.

In some embodiments, a hydrogel bead can be decreased in volume (e.g.,shrunken hydrogel bead) before being attached to or embedded in ahydrogel. In some embodiments, a hydrogel bead can be decreased involume (e.g., shrunken hydrogel bead) after being attached to orembedded in a hydrogel. In some embodiments, one or more hydrogel beadscan be attached to or embedded in a hydrogel. In some embodiments, oneor more hydrogel beads can be decreased in volume (e.g., one or moreshrunken hydrogel beads) before being attached to or embedded in ahydrogel. In some embodiments, one or more hydrogel beads can bedecreased in volume (e.g., one or more shrunken hydrogel beads) afterbeing attached to or embedded in a hydrogel. In some embodiments, one ormore hydrogel beads attached to or embedded in a hydrogel can bedecreased in volume. For example, the one or more hydrogel beads and thehydrogel that the hydrogel beads are attached to or embedded in aredecreased in volume at the same time (e.g., shrunken hydrogelbead-containing hydrogel). In some embodiments, one or more hydrogelbeads attached to or embedded in a hydrogel can be isometricallydecreased in volume.

In some embodiments, one or more hydrogel beads attached to or embeddedin a hydrogel can be decreased in volume from about 3 fold to about 4fold. For example, one or more hydrogel beads attached to or embedded ina hydrogel can be decreased in volume by removing or exchangingsolvents, salts, or water (e.g., dehydration). In another example, oneor more hydrogel beads attached to or embedded in a hydrogel can bedecreased in volume by controlling temperature or pH. See e.g., Ahmed,E.M. J. of Advanced Research. 2015 Mar;6(2):105-121, which isincorporated herein by reference in its entirety. In some embodiments,one or more hydrogel beads attached to or embedded in a hydrogel can bedecreased in volume by removing water.

In some embodiments, decreasing the volume of one or more hydrogel beadsattached to or embedded in a hydrogel can increase the spatialresolution of the subsequent analysis of the sample. The increasedresolution in spatial profiling can be determined by comparison of thespatial analysis of the sample using one or more shrunken hydrogel beadsattached to or embedded in a hydrogel with one or more non-shrunkenhydrogel beads attached to or embedded in a hydrogel.

In some embodiments, a hydrogel bead is not decreased in volume. In someembodiments, a hydrogel bead can be decreased in volume (e.g., shrunkenhydrogel bead). In some embodiments, a shrunken hydrogel gel bead isstabilized. For example, the hydrogel bead can be decreased in volume byremoving solvents, salts, or water (e.g., dehydrated, desiccated, dried,exsiccated) from the hydrogel bead to form a shrunken hydrogel bead. Inanother example, the hydrogel bead can be decreased in volume bycontrolling temperature or pH. See e.g., Ahmed, E.M. J. of AdvancedResearch. 2015 Mar;6(2):105-121, which is incorporated herein byreference in its entirety. Non-limiting examples of solvents that may beused to form a shrunken hydrogel bead or shrunken hydrogel bead arrayinclude a ketone, such as methyl ethyl ketone (MEK), isopropanol (IPA),acetone, 1-butanol, methanol (MeOH), dimethyl sulfoxide (DMSO),glycerol, propylene glycol, ethylene glycol, ethanol, (k) 1,4-dioxane,propylene carbonate, furfuryl alcohol, N,N-dimethylformamide (DMF),acetonitrile, aldehyde, such as formaldehyde or glutaraldehyde, or anycombinations thereof.

In some embodiments, the hydrogel bead or hydrogel bead array isshrunken or stabilized via a cross-linking agent. For example, thecross-linking agent may comprise disuccinimidyl suberate (DSS),dimethylsuberimidate (DMS), formalin, and dimethyladipimidate (DMA),dithio-bis(-succinimidyl propionate) (DSP), disuccinimidyl tartrate(DST), and ethylene glycol bis(succinimidyl succinate) (EGS).

In some embodiments, the hydrogel bead or hydrogel bead array isprocessed with salts to form a shrunken hydrogel bead or shrunkenhydrogel bead array. Non-limiting examples of salts that may be used toform a shrunken hydrogel bead or shrunken hydrogel bead array areinorganic salts including aluminum, ammonium, barium, beryllium,calcium, cesium, lithium, magnesium, potassium, rubidium, sodium, andstrontium salts. Further non-limiting examples of inorganic saltsinclude sodium chloride, potassium chloride, lithium chloride, cesiumchloride, sodium fluoride, sodium bromide, sodium iodide, sodiumnitrite, potassium sulfate, potassium nitrate, potassium carbonate,potassium bicarbonate, sodium sulfate, sodium nitrate, sodium carbonate,sodium bicarbonate, calcium sulfate, copper oxychloride, calciumchloride, calcium carbonate, calcium bicarbonate, magnesium sulfate,magnesium nitrate, magnesium chloride, magnesium carbonate, magnesiumbicarbonate, ammonium sulfate, ammonium chloride, ammonium nitrate,ammonium carbonate, ammonium bicarbonate, trisodium phosphate,tripotassium phosphate, calcium phosphate, copper(II) sulfate, sodiumsulfide, potassium sulfide, calcium sulfide, potassium permanganate,iron(II) chloride, iron(III) chloride, iron (2+) sulfate, iron(III)sulfate, iron(II) nitrate, iron(III) nitrate, manganese(II) chloride,manganese(III) chloride, manganese(II) sulfate, manganese(II) nitrate,zinc chloride, zinc nitrate, zinc sulfate, ammonium orthomolybdate,monopotassium phosphate, nickel(II) sulfate, nickel(II) nitrate, sodiummetavanadate, sodium paravanadate, potassium dichromate, ammoniumdichromate, antipyonin, ammonium nitrite, potassium fluoride, sodiumfluoride, ammonium fluoride, calcium fluoride, chrome alum, potassiumalum, potassium iodide, sodium hypochlorite, tin(II) sulfate, tin(II)nitrate, gold selenite, dicesium chromate, potassium perchlorate,calcium perchlorate, aluminum sulphate, lead(II) bisulfate, bariumphosphate, barium hydrogen orthophosphate, barium dihydrogen phosphate,silver dichromate, potassium bromate, sodium bromate, sodium iodate,sodium silicate, diammonium phosphate, ammonium phosphate, ammoniumdihydrogen phosphate, chromium orthophosphate, copper(II) chloride,copper(I) chloride, sodium tetrametaphosphate, potassiumheptafluoroniobate, zinc phosphate, sodium sulfite, copper(I) nitrate,copper(II) nitrate, potassium silicate, copper(II) carbonate basic,copper(II) carbonate salts of acrylic acid and sulfopropyl acrylate.

In some embodiments, the removal of water comprises an acid.Non-limiting examples of an acid include: HCl, HI, HBr, HClO4, HClO3,HNO3, H2SO4, phosphoric acid, phosphorous acid, acetic acid, oxalicacid, ascorbic acid, carbonic acid, sulfurous acid, tartaric acid,citric acid, malonic acid, phthalic acid, barbituric acid, cinnamicacid, glutaric acid, hexanoic acid, malic acid, folic acid, propionicacid, stearic acid, trifluoroacetic acid, acetylsalicylic acid, glutamicacid, azelaic acid, benzilic acid, fumaric acid, gluconic acid, lacticacid, oleic acid, propiolic acid, rosolic acid, tannic acid, uric acid,gallic acid, and combinations of two or more thereof. In someembodiments, the hydrogel is exposed to a different pH environment. Forexample, the hydrogel can be exposed to an acidic pH or a basic pH. Insome embodiments, the hydrogel is exposed to a pH of less than about6.5, e.g., a pH of about 6, about 5.5, about 5, about 4.5, about 4,about 3.5, about 3, about 2.5, about 2, about 1.5, or about 1. In someembodiments, the hydrogel is exposed to a pH of greater than about 7.5,e.g., a pH of about 8, about 8.5, about 9, about 9.5, about 10, about10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5,or about 14.

In some embodiments, the removal of water comprises a dehydratingprocess such as heat, a vacuum, lyophilization, desiccation, filtration,and air-drying. In some embodiments, the hydrogel bead or hydrogel beadarray undergoes an alteration in pH to form a shrunken hydrogel bead orshrunken hydrogel bead array (e.g., an alteration from about pH 7 toabout pH 5, from about pH 7 to about pH 5.5, from about pH 7 to about pH6, from about pH 7 to about pH 6.5, from about pH 6.5 to about pH 5,from about pH 6 to about pH 5, from about pH 6 to about pH 5.5, or anypH alteration encompassed within these ranges).

In some embodiments, the hydrogel bead or hydrogel bead array undergoesan alteration in temperature (e.g., an alteration from about 37° C., 38°C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47°C., 48° C., 49° C. to about 50° C., 51° C., 52° C., 53° C., 54° C., 55°C., 56° C., 57° C., 58° C., 59° C. 60° C., 61° C., 62° C., 63° C., 64°C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., or higher, or anytemperature alteration encompassed within these ranges) to form ashrunken hydrogel bead or shrunken hydrogel bead array.

In some embodiments, a hydrogel bead can be decreased in size in lineardimension by about 2 fold, about 3 fold, about 4 fold, about 5 fold,about 6 fold, about 7 fold, about 8 fold, about 9 fold, or any intervalstherein. In some embodiments, a hydrogel bead can be decreased in volumeby about 1 fold, about 5 fold, about 10 fold, about 15 fold, about 20fold, about 25 fold, about 30 fold, about 35 fold, about 40 fold, about45 fold, about 50 fold, about 55 fold, about 60 fold, about 65 foldabout 70 fold, about 75 fold, about 80 fold, or any intervals therein.In some embodiments, a hydrogel bead can be decreased in size such thatthe hydrogel bead has an average diameter of about 1 µm to about 15 µm.

In some embodiments, a plurality of hydrogel beads can be decreased insize in linear dimension by about 2 fold, about 3 fold, about 4 fold,about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, orany intervals therein. In some embodiments, a plurality of hydrogelbeads can be decreased in size such that the average diameter of ahydrogel bead is about 1 µm to about 15 µm. In some embodiments, aplurality of hydrogel beads can be decreased in volume by about 1 fold,about 5 fold, about 10 fold, about 15 fold, about 20 fold, about 25fold, about 30 fold, about 35 fold, about 40 fold, about 45 fold, about50 fold, about 55 fold, about 60 fold, about 65 fold about 70 fold,about 75 fold, about 80 fold, or any intervals therein.

In some embodiments, a plurality of hydrogel beads can be decreased involume such that the hydrogel bead has an average diameter of about 1 µmto about 15 µm. In some embodiments, a plurality of shrunken hydrogelbeads has an average diameter of about 15 µm. In some embodiments, aplurality of shrunken hydrogel beads has an average diameter of about 14µm. In some embodiments, a plurality of shrunken hydrogel beads has anaverage diameter of about 13 µm. In some embodiments, a plurality ofshrunken hydrogel beads has an average diameter of about 12 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 11 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 10 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 9 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 8 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 7 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 6 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 5 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 4 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 3 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 2 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 1 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 14-15 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 13-15 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 12-15 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 11-15 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 10-15 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 9-15 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 8-15 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 7-15 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 6-15 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 1-10 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 1-5 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 1-3 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 13-14 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 12-14 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 11-14 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 10-14 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 9-14 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 8-14 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 7-14 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 6-14 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 5-14 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 12-13 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 11-13 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 10-13 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 9-13 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 8-13 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 7-13 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 6-13 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 5-13 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 11-12 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 10-12 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 9-12 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 8-12 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 7-12 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 6-12 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 5-12 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 10-11 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 9-11 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 8-11 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 7-11 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 6-11 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 5-11 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 9-10 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 8-10 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 7-10 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 6-10 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 5-10 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 8-9 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 7-9 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 6-9 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 5-9 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 7-8 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 6-8 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 5-8 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 6-7 µm. In someembodiments, a plurality of shrunken hydrogel beads has an averagediameter of about 5-7 µm. In some embodiments, a plurality of shrunkenhydrogel beads has an average diameter of about 5-6 µm.

In some embodiments, one or more hydrogel beads can be decreased involume at the same time. In some embodiments, one or more hydrogel beadscan be decreased in volume at different times. In some embodiments, oneor more hydrogel beads can be assembled into an array before decreasingthe volume of the one or more hydrogel beads. In some embodiments, oneor more hydrogel beads can be assembled into an array after decreasingthe volume of the one or more hydrogel beads. In some embodiments, theone or more shrunken hydrogel beads can be reversibly attached to asubstrate. In some embodiments, the one or more shrunken hydrogel beadscan be irreversibly attached to a substrate. In some embodiments, theone or more shrunken hydrogel beads can be re-expanded. In someembodiments, the one or more shrunken hydrogel beads can beisometrically re-expanded. In some embodiments, the one or more shrunkenhydrogel beads can be re-expanded primarily in the z-dimension. In someembodiments, the one or more shrunken hydrogel beads attached to asubstrate (e.g., reversibly or irreversibly) can be re-expandedprimarily in the z-dimension. In some embodiments, the one or moreshrunken hydrogel beads attached to a substrate (e.g., reversibly orirreversibly) can be isometrically re-expanded primarily in thez-dimension.

In some embodiments, decreasing the volume of the hydrogel bead (e.g.,shrunken hydrogel bead) can increase the spatial resolution of thesubsequent analysis of the sample. The increased resolution in spatialprofiling can be determined by comparison of spatial analysis of thesample using a shrunken hydrogel bead with a non-shrunken hydrogel bead.For example, in some embodiments, the subsequent analysis of the samplecan include any array-based spatial analysis method disclosed herein.

In some embodiments, one or more physical parameters or dimensionsand/or one or more other characteristics of the hydrogel bead may bechanged. For example, a cross-section of the hydrogel bead may bechanged from a first cross-section to a second cross-section. The firstcross-section may be smaller or larger than the second cross-section.Alternatively, or in addition, one or more other characteristics of thehydrogel bead may be changed. For example, the fluidity, density,rigidity, porosity, refractive index, polarity, and/or othercharacteristic of the hydrogel bead or one or more components thereofmay be changed. In a non-limiting example, the hydrogel bead includes ahydrogel. In another example, the hydrogel bead hydrogel may formcrosslinks within the bead. The same or different conditions may be usedto change or affect different characteristics of the hydrogel bead atthe same or different times. In some cases, a first condition or set ofconditions may be used to change a first characteristic or set ofcharacteristics of the hydrogel bead (e.g., a cross-section) and asecond condition or set of conditions may be used to change a secondcharacteristic or set of characteristics of the hydrogel bead. The firstcondition or set of conditions may be applied at the same or a differenttime as the second condition or set of conditions. For example, a firstcharacteristic or set of characteristics may be changed under a firstcondition or set of conditions, after which a second characteristic orset of characteristics may be changed under a second condition or set ofconditions.

A characteristic or set of characteristics of the hydrogel bead may bechanged by one or more conditions. A condition suitable for changing acharacteristic or set of characteristics of the hydrogel bead may be,for example, a temperature, a pH, an ion or salt concentration, apressure, chemical species, any combinations thereof, or anothercondition. For example, hydrogel bead may be exposed to a chemicalspecies that may bring about a change in one or more characteristics ofthe hydrogel bead. In some cases, a stimulus may be used to change oneor more characteristics of the hydrogel bead. For example, uponapplication of the stimulus, one or more characteristics of the hydrogelbead may be changed. The stimulus may be, for example, a thermalstimulus, a photo stimulus, a chemical stimulus, or another stimulus. Atemperature sufficient for changing one or more characteristics of thehydrogel bead may be, for example, at least about 0° C. (°C), 1° C., 2°C., 3° C., 4° C., 5° C., 10° C., or higher. For example, the temperaturemay be about 4° C. In other cases, a temperature sufficient for changingone or more characteristics of the hydrogel bead may be, for example, atleast about 25° C., 30° C., 35° C., 37° C., 40° C., 45° C., 50° C., orhigher. For example, the temperature may be about 37° C. A pH sufficientfor changing one or more characteristics of the hydrogel bead may be,for example, between about 5 and 8, such as between about 6 and 7.

In some cases, a chemical species or a chemical stimulus may be used tochange one or more characteristics of the hydrogel bead. For example, achemical species or a chemical stimulus may be used to change adimension of a hydrogel bead (e.g., a cross-section, diameter, orvolume). In some cases, a chemical species or a chemical stimulus may beused to change a dimension of a hydrogel bead (e.g., a cross-sectionaldiameter) from a first dimension to a second dimension (e.g., a secondcross-sectional dimeter), where the second dimension is reduced comparedto the first dimension. The chemical species may comprise an organicsolvent, such as an alcohol, ketone, or aldehyde. For example, thechemical species may comprise acetone, methanol, ethanol, formaldehyde,or glutaraldehyde. The chemical species may comprise a cross-linkingagent. For example, the cross-linking agent may comprise disuccinimidylsuberate (DSS), dimethylsuberimidate (DMS), formalin, anddimethyladipimidate (DMA), dithio-bis(-succinimidyl propionate) (DSP),disuccinimidyl tartrate (DST), and ethylene glycol bis(succinimidylsuccinate) (EGS), and any combinations thereof. In some cases, across-linking agent may be a photo-cleavable cross-linking agent. Insome cases, a chemical stimulus may be used to change one or morecharacteristics of the hydrogel bead (e.g., a dimension of a hydrogelbead), where the chemical stimulus comprises one or more chemicalspecies. For example, the chemical stimulus may comprise a firstchemical species and a second chemical species, where the first chemicalspecies is an organic solvent and the second chemical species is across-linking agent. In some cases, a chemical stimulus may comprise achemical species that is a fixation agent that is capable of fixing orpreserving a hydrogel bead. For example, an organic solvent such as analcohol (e.g., ethanol or methanol), ketone (e.g., acetone), or aldehyde(e.g., formaldehyde or glutaraldehyde), or any combinations thereof mayact as a fixation agent. Alternatively, or in addition, a cross-linkingagent may act as a fixation agent. In some cases, a fixation agent maycomprise disuccinimidyl suberate (DSS), dimethylsuberimidate (DMS),formalin, and dimethyladipimidate (DMA), dithio-bis(-succinimidylpropionate) (DSP), disuccinimidyl tartrate (DST), and/or ethylene glycolbis(succinimidyl succinate) (EGS), and any combinations thereof. In somecases, a first chemical species and/or fixation agent may be provided toor brought into contact with the hydrogel bead to bring about a changein a first characteristic or set of characteristics of the hydrogelbead, and a second chemical species and/or fixation agent may beprovided to or brought into contact with the hydrogel bead to bringabout a change in a second characteristic or set of characteristics ofthe hydrogel bead. For example, a first chemical species and/or fixationagent may be provided to or brought into contact with the hydrogel beadto bring about a change in a dimension of a hydrogel bead (e.g., areduction in cross-sectional diameter), and a second chemical speciesand/or fixation agent may be provided to or brought into contact withthe hydrogel bead to bring about a change in a second characteristic orset of characteristics of the hydrogel bead (e.g., forming crosslinkswithin and/or surrounding the hydrogel bead). The first and secondchemical species and/or fixation agents may be provided to or broughtinto contact with the hydrogel bead at the same or different times.

In some embodiments, fixation may affect one or more parameters orcharacteristics of the hydrogel bead. For example, fixation may resultin shrinkage or volumetric reduction of the hydrogel bead. Fixation mayinclude dehydration of the hydrogel bead. Providing a fixation agent tothe hydrogel bead may result in a change in a dimension of the hydrogelbead. For example, providing a fixation agent to the hydrogel bead mayresult in a change in the volume or diameter of the hydrogel bead.Providing a fixation agent to the hydrogel bead may result in a changein a cross-section of the hydrogel bead (e.g., a cross-sectionaldiameter). For example, a first cross-section of the hydrogel bead priorto fixation may be different (e.g., larger) than a second cross-sectionof the hydrogel bead following fixation. In an example, an approximatelyspherical hydrogel bead may comprise a first cross section (e.g., across-sectional diameter) prior to fixation that is reduced in size to asecond cross-section following fixation. Providing a fixation agent tothe hydrogel bead may result in a second cross-section that is reducedby at least about 5% compared to the first cross-section. In some cases,the second cross-section may be reduced by at least 6%, 8%, 10%, 15%,25%, 30%, 35%, 40%, 45%, 50%, or more relative to the firstcross-section. For example, the second cross-section may be reduced byat least about 10%, 15%, 25%, or 50% relative to the firstcross-section. Fixation may also affect other features of the hydrogelbead. For example, fixation may result in a change in the porosity of amembrane or wall of a hydrogel bead, reorganization of components of thehydrogel bead, a change in hydrogel bead fluidity or rigidity, or otherchanges. In an example, a first fixation agent that is an organicsolvent is provided to the hydrogel bead to change a firstcharacteristic (e.g., hydrogel bead volume) and a second fixation agentthat is a cross-linking agent is provided to the hydrogel bead to changea second characteristic (e.g., hydrogel bead fluidity or rigidity). Thefirst fixation agent may be provided to the hydrogel bead before thesecond fixation agent.

In some instances, an approximately spherical hydrogel bead may comprisea first diameter prior to fixation (e.g., by an organic solvent) that isreduced in volume compared to a second diameter following fixation whenmaintained in a non-aqueous environment. Following fixation andreduction in volume to said second diameter, when maintained in anaqueous environment, the hydrogel bead may increase in volume to have adiameter substantially similar to the first diameter. In some cases, anapproximately spherical hydrogel bead may include a first diameter priorto fixation (e.g., by an organic solvent) that is reduced in volumecompared to a second diameter following fixation. Following fixation andreduction in volume to said second diameter, the hydrogel bead may becross-linked by a second fixative, wherein the second diameter issubstantially maintained in an aqueous environment followingcross-linking by the second fixative.

A change to a characteristic or set of characteristics of the hydrogelbead may be reversible or irreversible. In some cases, a change to acharacteristic or set of characteristics of the hydrogel bead may beirreversible, such that the change cannot be readily undone. Forexample, the volume, morphology, or other feature of the hydrogel beadmay be altered in a way that cannot be readily reversed. In an example,the change from a first cross-section of the hydrogel bead to a secondcross-section of the hydrogel bead is irreversible. In some cases, anirreversible change may be at least partially reversed upon theapplication of appropriate conditions and/or over a period of time. Inother cases, a change to a characteristic or set of characteristics ofthe hydrogel bead may be reversible. For example, the volume of ahydrogel bead may be reduced upon being subjected to a first conditionor set of conditions, and the volume of a hydrogel bead may be increasedto approximately the original volume upon being subjected to a secondcondition or set of conditions. Thus, the change from a firstcross-section of the hydrogel bead to the second cross-section may bereversible. A reversible change (e.g., a reversible volume reduction)may be useful in, for example, providing a hydrogel bead of a givenvolume to a given location, such as a partition. In some cases, a changeto a characteristic or set of characteristics of the hydrogel bead maybe only partially reversible. For example, the volume of a hydrogel beadmay be reduced (e.g., by dehydration), and the reduction in hydrogelbead volume may be accompanied by reorganization of components withinthe hydrogel bead. Upon reversal of the volume of the hydrogel bead(e.g., by rehydration), the arrangement of one or more components maynot revert to the original arrangement of the hydrogel bead prior to thevolume reduction. A change to a characteristic or set of characteristicsof the hydrogel bead, such as a cross-section of the hydrogel bead, maybe reversible upon application of a stimulus. The stimulus may be, forexample, a thermal stimulus, a photo stimulus, or a chemical stimulus.In some cases, the stimulus may comprise a change in pH and/orapplication of a reducing agent such as dithiothreitol. Application ofthe stimulus may reverse, wholly or in part, a change from, for example,a first cross-section to a second cross-section.

In some embodiments, a plurality of hydrogel beads can be shrunkenhydrogel beads generated by removing water from a plurality of firsthydrogel beads. In some embodiments, the plurality of shrunken hydrogelbeads has an average diameter no larger than about 15 microns. Forexample, the plurality of shrunken hydrogel beads has an averagediameter no larger than about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,3, 2, or 1 micron. In some embodiments, each member of the plurality ofshrunken hydrogel beads has a diameter no larger than about 15 microns.For example, each member of the plurality of shrunken hydrogel beads canhave a diameter no larger than about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,5, 4, 3, 2, or 1 micron. In some embodiments, the plurality of shrunkenhydrogel beads has an average diameter no larger than 10 microns. Insome embodiments, each member of the plurality of shrunken hydrogelbeads has a diameter no larger than 10 microns. In some embodiments, theplurality of shrunken hydrogel beads has an average diameter no largerthan 5 microns. In some embodiments, each member of the plurality ofshrunken hydrogel beads has a diameter no larger than 5 microns. In someembodiments, the plurality of shrunken hydrogel beads has an averagediameter no larger than 1 micron. In some embodiments, each member ofthe plurality of shrunken hydrogel beads has a diameter no larger than 1micron. In some embodiments, the plurality of shrunken hydrogel beadshas an average diameter no larger than the diameter of a cell (e.g., amammalian cell, a plant cell, or a fungal cell). In some embodiments,each member of the plurality of shrunken hydrogel beads has a diameterno larger than the diameter of a cell (e.g., a mammalian cell, a plantcell, or a fungal cell).

In some embodiments, the plurality of shrunken hydrogel beads has apolydispersity index of less than about 25%. For example, the pluralityof shrunken hydrogel beads can have a polydispersity index of less thanabout 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In someembodiments, the plurality of shrunken hydrogel beads has apolydispersity index of less than 15%. In some embodiments, theplurality of shrunken hydrogel beads has an average diameter of about 8to about 13 microns. In some embodiments, the plurality of shrunkenhydrogel beads has an average diameter of about 10 to about 12 microns.In some embodiments, the plurality of shrunken hydrogel beads has anaverage diameter of about the diameter of a cell (e.g., a mammaliancell, a plant cell, or a fungal cell). In some embodiments, theplurality of shrunken hydrogel beads has an average diameter of lessthan the diameter of a cell (e.g., a mammalian cell, a plant cell, or afungal cell). In some embodiments, the plurality of capture probes onthe plurality of shrunken gel beads bind cellular analytes atsingle-cell resolution. In some embodiments, the plurality of captureprobes on the plurality of shrunken gel beads bind cellular analytes athigher than single-cell resolution (e.g., at a resolution that is at ahigher density than the diameter of a cell).

In some embodiments, bead arrays having a plurality of hydrogel beadsdisposed on a substrate are generated by patterning or self-assembly oflarger gel beads, after which the array of larger gel beads is shrunken(e.g., by any of the variety of methods provided herein). In someembodiments, the larger gel beads are not small enough for single-cellresolution, while the shrunken gel beads are small enough forsingle-cell resolution. In some embodiments, bead arrays having aplurality of hydrogel beads disposed on a substrate are generated bypatterning or self-assembly of shrunken gel beads that have previouslybeen generated by shrinking larger gel beads (e.g., by any of thevariety of methods provided herein). Beads can be spatially confined byany of a variety of methods, including without limitation, reversible orirreversible crosslinking.

In some embodiments, bead arrays include spatially-confined gel beadswith high aspect ratios (e.g., pillared arrays). For example, beadarrays having a plurality of hydrogel beads disposed on a substrate canbe generated by any of the variety of methods described herein (e.g., bypatterning or self-assembly of shrunken gel beads or by patterning orself-assembly of larger gel beads followed by shrinking), after whichthe high-density bead array is expanded (or re-expanded). Whenexpanding, spatial constraints direct the beads to expand primarily inthe Z dimension (away from the substrate), resulting in pillar arrays.In some embodiments, the gel beads of the pillar arrays have high aspectratios. In some embodiments, aspect ratio of the expanded plurality ofspatially-confined shrunken hydrogel beads is at least 2. In otherembodiments, the aspect ratio of the expanded plurality ofspatially-confined shrunken hydrogel beads is at least 3. In someembodiments, the plurality of spatially-confined shrunken hydrogel beadshas an average aspect ratio of at least 4, 5, 6, 7, 8 or more.

In some embodiments, the method for the removal of water from a hydrogelis the same for each hydrogel (e.g., the first hydrogel, the secondhydrogel, or the third hydrogel). In some embodiments, the method forthe removal of water from one hydrogel (e.g., the first hydrogel) isdifferent from the method for the removal of water for at least oneother hydrogel (e.g., a second hydrogel, a third hydrogel, or a fourthhydrogel). For example, the method for the removal of water from onehydrogel can be different from the method for the removal of water forthe other hydrogels (e.g., a second hydrogel, a third hydrogel, or afourth hydrogel). In some embodiments, the method for the removal ofwater is different for each hydrogel (e.g., the first hydrogel, thesecond hydrogel, the third hydrogel, and the fourth hydrogel).

In some embodiments, the shrunken hydrogel is at least about 2-foldsmaller in a linear dimension (e.g., along one axis) than the pre-shrunkhydrogel. For example, at least about 2.5, about 3, about 4, about 5,about 6, about 7, about 8, about 9, about 10, or more fold smaller in alinear dimension than the pre-shrunk hydrogel.

In some embodiments, the size of the hydrogel is reduced along more thanone axes, e.g., along 2 or 3 axes. In some embodiments, each axisintersects each other axis at 90 degrees. In some embodiments, the sizeof the hydrogel along the first axis is about 2 to about 10 or more foldsmaller than the pre-shrunk hydrogel, e.g., about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10 or more foldsmaller than the pre-shrunk hydrogel. In some embodiments, the size ofthe hydrogel along the second axis is about 2 to about 10 or more foldsmaller than the pre-shrunk hydrogel, e.g., about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10 or more foldsmaller than the pre-shrunk hydrogel. In some embodiments, the size ofthe hydrogel along the third axis is about 2 to about 10 or more foldsmaller than the pre-shrunk hydrogel, e.g., about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10 or more foldsmaller than the pre-shrunk hydrogel. In some embodiments, the reductionin the volume of the hydrogel is isometric.

In some embodiments, the volume of each hydrogel (e.g., a firsthydrogel, a second hydrogel, a third hydrogel, or a fourth hydrogel) isthe same. In some embodiments, the volume of at least one hydrogel isdifferent. For example, in some embodiments, one hydrogel is differentin volume from the other hydrogels (e.g., a second hydrogel, a thirdhydrogel, or a fourth hydrogel). In some embodiments, every hydrogel isdifferent in volume from every other hydrogel.

In some embodiments, members of the plurality of features arecross-linked to a hydrogel (e.g., a first hydrogel, a second hydrogel, athird hydrogel, or a fourth hydrogel).

In one embodiment, features of an array can be copied into a hydrogel,and the volume of the hydrogel is reduced by removing water. These stepscan be performed multiple times. For example, a method for preparing ahigh-density spatially-barcoded flexible array can include copying aplurality of spatially-barcoded features from an array into a firsthydrogel, wherein the first hydrogel is in contact with the array;reducing the volume of the first hydrogel including the copied featuresby removing water, forming a first shrunken hydrogel including thecopied features; copying the features in the first shrunken hydrogelinto a second hydrogel, where the second hydrogel is in contact with thefirst hydrogel; and reducing the volume of the second hydrogel includingthe copied features by removing water, forming a second shrunkenhydrogel including the copied features, thus generating a high-densityspatially-barcoded array. The process of copying spatially-barcodedfeatures from an array to a first hydrogel, removing water from thefirst hydrogel to form a first shrunken hydrogel, and copyingspatially-barcoded features from the first shrunken hydrogel to one ormore subsequent hydrogels can be performed multiple times (e.g., 2, 3,4, 5, 6, 7, 8, 9, or 10 times). The result is a high-density flexiblearray including spatially-barcoded features.

In some embodiments, copying members of the plurality of features froman array includes copy by PCR. In some embodiments, the hydrogel (e.g.,a first hydrogel, a second hydrogel, a third hydrogel, and/or a fourthhydrogel) comprises PCR reagents as described herein. In someembodiments, members of the plurality of features are copied usingreplica plating techniques (see, e.g., Mitra and Church, Nucleic AcidsRes. 1999 Dec 15;27(24):e34, which is incorporated by reference hereinin its entirety). In some embodiments, after copying a plurality offeatures from an array into a first hydrogel, the features of the arrayare amplified in the first hydrogel (e.g., clonal amplification). Insome embodiments, members of the plurality of features are copied intothe first hydrogel such that the pattern of the plurality of features ofthe first hydrogel is the same or substantially similar (e.g., at least80%) to the pattern of the plurality of features of the array.

In some embodiments, one or more pluralities of features of the arrayare partitioned. For example, each partition can comprise a plurality offeatures different from the plurality of features of other partitions.For example, the members of the plurality of features are partitionedsimilar to the partitions of the plurality of features of the array. Insome embodiments, the features of the array are copied into the firsthydrogel, such that the volume or diameter of the pre-shrunk firsthydrogel features are similar to the volume or diameter of the arrayfeatures.

In some embodiments, the volume of a hydrogel comprising copied featuresis reduced, thus increasing the density of the copied features. In someembodiments, the copied features within a hydrogel further increases indensity with each subsequent hydrogel copy and shrinking. For example,the density of the copied features of a second shrunken hydrogel ishigher than the density of the copied features of a first shrunkenhydrogel. Similarly, the density of the copied features of a thirdshrunken hydrogel is higher than the density of the copied features of asecond shrunken hydrogel. Similarly, the density of the copied featuresof a fourth shrunken hydrogel is higher than the density of the copiedfeatures of a third shrunken hydrogel. In some embodiments, the volumeof a partition of members of the plurality of features in a hydrogel isreduced when the volume of the hydrogel is reduced.

In some embodiments of the methods described herein, an array comprisesshrunken gel features (e.g., beads). In some embodiments, the methodsdescribed herein generate shrunken gel bead arrays. In some embodiments,the shrunken gel beads of the array are shrunken hydrogel beads.

A “shrunken array” includes a plurality of spatially-barcoded featuresattached to, or embedded in, a substrate that have been reduced involume (e.g., reduction in diameter or volume). A biological sample canbe contacted with a shrunken array and further contacted with a solutioncapable of rehydrating the shrunken array. In some embodiments, analytetransfer and capture is driven by molecular diffusion. The process ofrehydrating the shrunken array by providing a permeabilization solutionor tissue stain to the sample can promote analytes (e.g., transcripts)present in the biological sample towards the spatially-barcodedfeatures, thereby improving capture efficiency of the analytes. See,e.g., J. Vlassakis, A. E. Herr. “Effect of Polymer Hydration State onIn-Gel Immunoassays.” Anal. Chem. 2015, 87(21):11030-8, hereinincorporated by reference in its entirety.

A shrunken array can be generated with features (e.g., beads) containingspatial barcodes from an existing array. For example, an array (e.g.,hydrogel bead array) described and prepared by any method herein can becontacted with reagents capable of dehydrating (e.g., removing water)the features (e.g., beads) to generate a shrunken array (e.g., ashrunken bead array). Methods of dehydrating features (e.g., beads) areknown in the art. Any suitable method of dehydration (e.g., removingwater) can be used. For example, in a non-limiting way, features (e.g.,beads) can be dehydrated by a ketone, such as methyl ethyl ketone (MEK),isopropanol (IPA), acetone, 1-butanol, methanol (MeOH), dimethylsulfoxide (DMSO), glycerol, propylene glycol, ethylene glycol, ethanol,(k) 1,4-dioxane, propylene carbonate, furfuryl alcohol,N,N-dimethylformamide (DMF), acetonitrile, aldehyde, such asformaldehyde or glutaraldehyde, or any combinations thereof. Additionaldehydration agents include various salts, including inorganic salts(See, e.g., Ahmed, E.M., Hydrogel: Preparation, characterization, andapplications: A review, Journal of Advanced Research, 6 (2) 105-121(2015), which is incorporated herein by reference).

In some embodiments, the dehydrated features (e.g., beads) can create ashrunken array (e.g., shrunken bead array or shrunken hydrogel array)where the average diameter of the dehydrated features (e.g., beads) canbe smaller than the average diameter of the features prior todehydration. In some embodiments, the dehydrated features (e.g., beads)can have an average diameter at least two-fold smaller than the averagediameter of the features prior to dehydration. In some embodiments, thedehydrated features (e.g., beads) can have an average diameter at leastthree-fold smaller than the average diameter of the features prior todehydration. In some embodiments, the dehydrated features (e.g., beads)can have an average diameter at least four-fold or smaller than theaverage diameter of the features (e.g., beads) prior to dehydration.

After generating a shrunken array, a biological sample (e.g., tissuesample) can be contacted with the shrunken array (e.g., shrunken beadarray). A rehydrating solution can be provided to the biological sampleand the shrunken array by any suitable method (e.g., by pipetting). Therehydrating solution can contain reagents to rehydrate (e.g., water orbuffers) the features (e.g., beads) of the shrunken array. In someembodiments, the rehydrating solution can be applied to the entirebiological sample. In some embodiments, the rehydrating solution can beselectively applied (e.g., to a region of interest). In someembodiments, absorbing water from the rehydrating solution can increasethe diameter of at least one feature (e.g., bead) in the shrunken array.In some embodiments, the rehydrating solution can increase the diameterof at least one feature (e.g., bead) by at least two-fold. In someembodiments, the rehydrating solution can increase the diameter of atleast one feature (e.g., bead) by at least three-fold. In someembodiments, the rehydrating solution can increase the diameter of atleast one feature (e.g., bead) by at least four-fold. In someembodiments, the rehydrating solution can increase the diameter of atleast one feature (e.g., bead) by at least five-fold or more.

In some embodiments, the rehydrating solution can containpermeabilization reagents. The biological sample can be permeabilizedusing permeabilization reagents and techniques known in the art orotherwise described herein. Biological samples from different sources(e.g., brain, liver, ovaries, kidney, breast, colon, etc.) can requiredifferent permeabilization treatments. For example, permeabilizing thebiological sample (e.g., using a protease) can facilitate the migrationof analytes to the substrate surface (e.g., spatially-barcodedfeatures). In some embodiments, the permeabilization reagents can be adetergent (e.g., saponin, Triton X-100™, Tween-20™). In someembodiments, an organic solvent (e.g., methanol, acetone) canpermeabilize cells of the biological sample. In some embodiments, anenzyme (e.g., trypsin) can permeabilize the biological sample. Inanother embodiment, an enzyme (e.g., collagenase) can permeabilize thebiological sample.

In some embodiments the solution can permeabilize the biological sampleand rehydrate the features (e.g., beads) of the shrunken array (e.g.,shrunken hydrogel). In some embodiments, the rehydrating solution canstain the biological sample and rehydrate the features of the shrunkenarray (e.g., beads).

In some embodiments, the rehydrating solution (e.g., permeabilization orstain solution) can diffuse through the biological sample. In someembodiments, the rehydrating solution can reduce diffusion of analytesaway from the substrate. In some embodiments, while diffusing throughthe biological sample, the rehydrating solution can migrate analytestoward the substrate surface and improve the efficiency of analytecapture.

(VIII) Microcapillary Arrays

A “microcapillary array” is an arrayed series of features that arepartitioned by microcapillaries. A “microcapillary channel” is anindividual partition created by the microcapillaries. For example,microcapillary channels can be fluidically isolated from othermicrocapillary channels, such that fluid or other contents in onemicrocapillary channel in the array are separated from fluid or othercontents in a neighboring microcapillary channel in the array. Thedensity and order of the microcapillary channels can be any suitabledensity or order of discrete sites.

In some embodiments, microcapillary arrays are treated to generateconditions that facilitate loading. An example is the use of a coronawand (BD-20AC, Electro Technic Products) to generate a hydrophilicsurface. In some embodiments, a feature (e.g., a bead with captureprobes attached) is loaded onto a microcapillary array such that theexact position of the feature within the array is known. For example, acapture probe containing a spatial barcode can be placed into amicrocapillary channel so that the spatial barcode can enableidentification of the location from which the barcoded nucleic acidmolecule was derived.

In some embodiments, when random distribution is used to distributefeatures, empirical testing can be performed to generateloading/distribution conditions that facilitate a single feature permicrocapillary. In some embodiments, it can be desirable to achievedistribution conditions that facilitate only a single feature (e.g.,bead) per microcapillary channel. In some embodiments, it can bedesirable to achieve distribution conditions that facilitate more thanone feature (e.g., bead) per microcapillary channel, by flowing thefeatures through the microcapillary channel.

In some embodiments, the microcapillary array is placed in contact witha sample (e.g., on top or below) so that microcapillaries containing afeature (e.g., a bead, which can include a capture probe) are in contactwith the biological sample. In some embodiments, a biological sample isplaced onto an exposed side of a microcapillary array and mechanicalcompression is applied, moving the biological sample into themicrocapillary channel to create a fluidically isolated reaction chambercontaining the biological sample.

In some embodiments, a biological sample is partitioned by contacting amicrocapillary array to the biological sample, thereby creatingmicrocapillary channels including a bead and a portion of the biologicalsample. In some embodiments, a portion of a biological sample containedin a microcapillary channel is one or more cells. In some embodiments, afeature is introduced into a microcapillary array by flow after one ormore cells are added to a microcapillary channel.

In some embodiments, reagents are added to the microcapillary array. Thereagents can include enzymatic reagents or reagent mixtures forperforming amplification of a nucleic acid. In some embodiments, thereagents include a reverse transcriptase, a ligase, one or morenucleotides, or any combinations thereof. One or more microcapillarychannels can be sealed after reagents are added to the microcapillarychannels, e.g., by using silicone oil, mineral oil, a non-porousmaterial, or lid.

In some embodiments, a reagent solution is removed from eachmicrocapillary channel following an incubation for an amount of time andat a certain temperature or range of temperatures, e.g., following ahybridization or an amplification reaction. Reagent solutions can beprocessed individually for sequencing, or pooled for sequencinganalysis.

(IX) Hydrogel / Well Arrays

In some embodiments are methods for generating patterned hydrogel arraysusing wells (e.g., a nanowell or microwell array). In some embodiments,the well is a 3-dimensional structure. In some embodiments, the top viewof a well is any suitable 2-dimensional shape, which when extended alongthe z-axis, produces a 3-dimensional structure capable of containing oneor more features (e.g., beads) and/or reagents. Non-limiting examples ofwells which may form an array include a triangular prism, a square orrectangular prism, a pentagonal prism, a hexagonal prism, a heptagonalprism, an octagonal prism, an n-sided prism, or a cylindrical array(e.g., “microcapillary array”). In some embodiments, a well of the wellarray shares at least one well wall (or a portion of the well wall, if amicrocapillary array) with an adjacent well. In some embodiments, a welldoes not share any walls or portion of a wall in common with anotherwell of the array. In some embodiments, the well array is attached to asubstrate, such that the wells of the well array are fluidicallyisolated from each other. In some embodiments, one end of the well arrayis open (e.g., exposed), wherein the open end can be used to distributefeatures or reagents into the well.

In some embodiments, the method includes providing shrunken (e.g.,dehydrated) hydrogel features (e.g., beads) to a well array. Thehydrogel features can be dehydrated (e.g., removing water) by any of thevariety of methods described herein. The features, described elsewhereherein, can be provided such that the number of features is less thanthe number of wells of the array, the features can be provided such thatthe number of features equals the number of wells of the array, or thefeatures can be provided in excess of the number of wells of the array.In some embodiments, the well array is manipulated such that one or moreshrunken hydrogel features move from the top surface of the array downinto a well. For example, a well array can be placed on a shaker for alength of time necessary for the features to distribute into the wells.Other, non-limiting examples of manipulations that can cause a shrunkenhydrogel feature to enter a well are physically shaking, tilting, orrolling the well array, or a combination thereof; using forced air toblow features into a well, using a magnet to pull down hydrogel featurescomprising magnetic particles, using microfluidic systems to distributefeatures into wells, using a printer to deposit a feature into a well,or any other method to distribute features into wells. In someembodiments, once a well contains a feature, the well cannot accept orretain another feature. In other embodiments, a well can contain morethan one feature.

In some embodiments, the method includes rehydrating (e.g., addingwater) the shrunken hydrogel features, wherein the shrunken hydrogelfeatures are located in the wells. Rehydrating shrunken hydrogelfeatures can be accomplished by any method described herein. Rehydratinga shrunken hydrogel feature in the well can cause the shrunken hydrogelfeature to expand. In some embodiments, the shrunken hydrogel featureexpands to fill the well. In some embodiments, the shrunken hydrogelfeature expands in a z direction, such that the feature expands out ofthe unenclosed (i.e. open) end of the well. The exposed area of therehydrated feature can create a patterned hydrogel array (e.g., a wellarray). A rehydrated feature contained within a well can be stable. Insome embodiments, a rehydrated feature (e.g., a rehydrated shrunkenhydrogel feature) is immobilized within a well, such that typical arrayusage does not dissociate the rehydrated feature from the well. Thepatterned hydrogel array can be used for analyte capture according tothe methods described herein.

(X) Bead Tethering

“Bead tethering” can refer to an arrangement of beads, wherein thearrangement may or may not form an array. The tethered beads can becontacted with a sample and processed according to methods describedherein. Further, contacting a biological sample with a single bead orbeads tethered together in various arrangements can allow for moreprecise spatial detection of analytes, e.g., a region of interest.Methods for tethering beads together are known in the art. Somesuitable, non-limiting, methods of tethering beads together can be,e.g., chemical linkers, proximity ligation, or any other methoddescribed herein. In some embodiments, beads can be tethered togetherindependent of a substrate. In some embodiments, beads can be tetheredin various arrangements on an existing substrate. In some embodiments, asubstrate (e.g., a hydrogel) can be formed around existing tetheredbeads. In some embodiments, the beads or bead arrangement can contact aportion of the biological sample. In some embodiments, the bead or beadarrangement can contact a region of interest. In some embodiments, thebeads or bead arrangement can contact the entire biological sample. Insome embodiments, the beads or bead arrangements are contacted to randompositions on the biological sample. In some embodiments, the beads arecontacted to according to a specific pattern on the biological sample.

Beads can be tethered together in various arrangements. In someembodiments, a single (e.g., one) bead can be contacted with abiological sample. In some embodiments, two or more beads can betethered (e.g., connected to each other), in various arrangements. Forexample, in a non-limiting way, beads can be tethered together to form acluster, a row, or arranged on a mesh (e.g., a net). In someembodiments, at least three beads can be tethered together in atwo-dimensional (2D) array (e.g., a cluster). In some embodiments, atleast two beads can be tethered together in a one-dimensional (1D) array(e.g., a row). In such embodiments, the beads are arranged in suchfashion that the beads can contact each other directly. In someembodiments, at least two beads can be tethered together in a stringarrangement. In such embodiments, the beads are arranged in such fashionthat the beads can contact each other indirectly (e.g., beads areconnected via linker). In some embodiments, at least two beads can betethered together in a mesh arrangement (e.g., net). In someembodiments, beads tethered together in a 2D array, a 1D array, thebeads on a string arrangement, and the beads on the mesh arrangement canbe used in any combination with each other on the biological sample.

In some embodiments, at least about 2 to about 10 beads can be tetheredtogether in various arrangements. In some embodiments at least about 3,about 4, about 5, about 6, about 7, about 8, about 9, or more beads canbe tethered together. In some embodiments, about 10 to about 100 beadscan be tethered together in various arrangements. In some embodiments,about 10, about 20, about 30, about 40, about 50, about 60, about 70,about 80, about 90, or more beads can be tethered together in variousarrangements. In some embodiments, about 100 to about 1,000 beads can betethered together in various arrangements. In some embodiments, about100, about 200, about 300, about 400, about 500, about 600, about 700,about 800, or about 900 or more beads can be tethered together invarious arrangements. In some embodiments, about 1000 to about 10000beads can be tethered together in various arrangements. In someembodiments, about 1000, about 2000, about 3000, about 4000, about 5000,about 6000, about 7000, about 8000, about 9000, about 10000 or morebeads can be tethered together.

In some embodiments, the tethered beads can have capture probescomprising spatial barcodes, functional domains, unique molecularidentifiers, cleavage domains, and capture domains, or combinationsthereof. In some embodiments, each bead can be associated with a uniquespatial barcode. In some embodiments, the spatial barcode is known priorto contacting the bead or bead arrangement to the biological sample. Insome embodiments, the spatial barcode is not known prior to contactingthe bead or bead arrangement to the biological sample. The identity ofeach bead (e.g., spatial barcode) in the array can be deconvolved, forexample, by direct optical sequencing, as discussed herein.

(XI) Printing Arrays in Liquid

In some embodiments, an array can be printed in liquid. The resolutionof conventionally-printed arrays can be limited, due to the diffusion ofprinted solutions. Printing the array in a highly viscous liquid canincrease resolution by preventing the diffusion of the printed solution.Thus, disclosed herein are various methods and materials for attachingand/or introducing a capture probe (e.g., a nucleic acid capture probe)having a barcode (e.g., a spatial barcode) to a substrate (e.g., aslide), wherein the attaching (e.g., printing) is performed in liquid.

In some aspects, capture probes are printed on a substrate (e.g., aslide or bead). In some aspects, the substrate is a slide. In someaspects, the substrate is a 96-well microtiter plate. In some aspects,methods provided herein can also be applied to other substrates commonlyused for nucleic acid analyses, including but not limited to beads,particles, membranes, filters, dipsticks, slides, plates, andmicrochips. In some aspects, such substrates may be composed of a numberof materials known to be compatible with nucleic acid analysis,including but not limited to agarose, styrene, nylon, glass, andsilicon.

First Solution

In some embodiments, provided herein are methods of printing arrays onsubstrates using one or more liquid solution(s) (e.g., two or moresolutions that include distinct capture probes). In some aspects,methods of printing arrays on substrates using one or more solution(s)can improve the resolution of the printed array. In some aspects,methods provided herein include dispensing a first solution (e.g., bulksolution) onto a substrate. In some aspects, the first solution (e.g.,bulk solution) has a lower Reynolds Number relative to a second solution(e.g., a second solution that includes capture probes to be attached tothe substrate). The Reynolds Number represents an inverse relationshipbetween the density and velocity of a fluid and its viscosity in achannel of given length. More viscous, less dense, and/or slower movingfluids will have a lower Reynolds Number, and are easier to divert,stop, start, or reverse without turbulence. In some embodiments, thefirst solution and the second solution are immiscible.

In some aspects, the first (e.g., bulk) solution is hydrophobic. In someaspects, after dispensing the first (e.g., bulk) solution onto theslide, the first (e.g., bulk) solution remains on the slide in discretespatial areas on the slide. In some aspects, the first (e.g., bulk)solution is made of a solution that does not denature one or more probesand/or does not inhibit probe-to-substrate binding. In some embodiments,the bulk solution can include an aqueous solution, a high viscositysolution, or a low nucleic acid diffusivity solution. In some aspects,the first (e.g., bulk) solution is a gel. In some aspects, the first(e.g., bulk) solution is a hydrogel. In some aspects, the first (e.g.,bulk) solution includes natural polymers, including for example,glycerol, collagen, gelatin, sugars such as starch, alginate, andagarose, or any combinations thereof. In some aspects, the first (e.g.,bulk) solution includes a synthetic polymer. In some aspects, thesynthetic polymer is prepared by any method known in the art, includingfor example, chemical polymerization methods. In some aspects, the gelor polymer is hydrophobic. In some aspects, the gel or polymer ishydrophilic. In some aspects, the gel or polymer is aqueous. In someaspects, the gel or polymer shrinks at room temperature. In someaspects, the gel or polymer shrinks when heated. In some aspects, thepolymer is a film that shrinks when heated.

In some aspects, the first (e.g., bulk) solution includes glycerol. Insome aspects, glycerol is present in the first (e.g., bulk) solution ata concentration of 5-100%. In some aspects, glycerol is present in thesolution at a concentration of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, or 70%.

In some aspects, the first (e.g., bulk) solution includes sugar. In someaspects, the sugar is a monosaccharide, a disaccharide, apolysaccharide, or combinations thereof. In some aspects, the sugar isglucose, fructose, mannose, galactose, ribose, sorbose, ribulose,lactose, maltose, sucrose, raffinose, starch cellulose, or combinationsthereof. In some aspects, sugar is sourced from complex compounds suchas molasses or other by-products from sugar refinement. In some aspects,sugar is present in the first (e.g., bulk) solution at a concentrationof 5-100%. In some aspects, sugar is present in the solution at aconcentration of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, or 70%.

In some aspects, the first (e.g., bulk) solution has a viscosity that isabout 0.1x-fold, 0.2x-fold, 0.3x-fold, 0.4x-fold, 0.5x-fold, 0.6x-fold,0.7x-fold, 0.8x-fold, 0.9x-fold, 1.0x-fold, 1.1x-fold, 1.2x-fold,1.3x-fold, 1.4-fold, 1.5x-fold, 1.6x-fold, 1.7x-fold, 1.8x-fold,1.9x-fold, 2.0x-fold, 2.5x-fold, 3.0x-fold, 4.0x-fold, 5.0x fold,6.0x-fold, 7.0x-fold, 8.0x fold, 9.0x-fold, or 10x-fold greater than theviscosity of the second solution.

Second Solution

In some embodiments, printing arrays on substrates using two or moresolutions includes using a second solution. In some embodiments, thesecond solution can include one or more capture probes. In someembodiments, the second solution is dispensed in the form of a droplet.Some embodiments include a plurality of second solutions, wherein eachmember of the plurality includes one or more capture probes comprising aspatial barcode unique to that particular droplet. In some embodiments,the second solution is dispensed onto a substrate covered or partiallycovered with the first (e.g., bulk) solution. In some embodiments, thefirst (e.g., bulk) solution reduces or prevents the diffusion of one ormore capture probes from the second solution. In some aspects, the oneor more capture probes remain entirely within the second solution whenprinted onto a substrate covered or partially covered with the first(e.g., bulk) solution.

In some aspects, the second solution includes one or more capture probes(e.g., any of the capture probes disclosed herein). In some aspects, thesecond solution includes one spatially-barcoded capture probe. In someaspects the second solution includes at least 5, at least 10, at least20, at least 30 at least 40, at least 50, at least 75, at least 100, atleast 200, at least 300, at least 400, at least 500, at least 600, atleast 700, at least 800, at least 900, at least 1,000, at least 1,500,at least 2,000, at least 3,000, at least 4,000, or at least 5,000spatially-barcoded capture probes. In some aspects, the second solutionincludes compounds that facilitate binding of the one or more captureprobes to the substrate. In some aspects, the second solution does notinhibit the one or more capture probes from binding the substrate and/ordoes not denature the one or more capture probes. In some aspects, thesecond solution is less viscous than the first (e.g., bulk) solution. Insome aspects, the second solution and the first (e.g., bulk) solutionare wholly or substantially immiscible (e.g., they do not mix). In someaspects, the second solution is an aqueous solution. In some aspects,the second solution is a hydrophilic solution.

Dispensing

In some embodiments, spot printing a high-density pattern of featurescan include dispensing the oligonucleotides and/or features, in the formof a liquid droplet, onto the surface of the substrate in the presenceof a bulk solution. In some embodiments, the second solution droplet(s)(e.g., oligonucleotides and/or feature droplet(s)) and the first (e.g.,bulk) solution does not substantially mix with each other. In someaspects, the printing methods disclosed herein that include dispensing asecond solution including one or more spatially-barcoded capture probesonto a substrate in the presence (e.g., through) a first (e.g., bulk)solution result in a spot size of the second solution (e.g., across-sectional spot size of the second solution on the plane of thesubstrate) that is smaller compared to the spot size that would beobtained by dispensing the same second solution onto the substrate inthe absence of the first (e.g., bulk) solution. In some aspects, thespot size (e.g., the cross-sectional spot size of the second solution onthe plane of the substrate) of the second solution does not increaseafter printing the second solution onto the substrate in the presence ofthe first (e.g., bulk) solution. In some aspects, the area of the spotof the second solution does not change after printing the second ontothe substrate in the presence of the first (e.g., bulk) solution. Insome aspects, printing of the second solution onto the substrate in thepresence of the first (e.g., bulk) solution results in a desired patternon the substrate surface. For example, a plurality of second solutionscan be printed onto the substrate in the presence of the first solutionsuch that the locations where the plurality of second solutions areprinted results in a desired pattern on the substrate. In someembodiments, two or more members of a plurality of second solutionsprinted onto a substrate include distinct populations of capture probes,which capture probes attach to the substrate, such that an array ofcapture probes is generated.

Density

In some embodiments, the cross-sectional area of the oligonucleotideand/or feature droplet(s) on the substrate is smaller than acorresponding cross-sectional area of the oligonucleotide and/or featuredroplet(s) that would be generated by dispensing the oligonucleotidesand/or features onto the surface of the substrate in the absence of thebulk solution. In some embodiments, the cross-sectional area of theoligonucleotide and/or feature droplet(s) on the substrate is abouttwo-fold smaller than the corresponding cross-sectional area of theoligonucleotide and/or feature droplet(s) that would be generated bydispensing the oligonucleotide and/or feature droplet(s) onto thesurface of the substrate in the absence of the bulk solution.

In some aspects, the cross-sectional area of the second solution on thesubstrate generated by dispensing the second solution onto the surfaceof the substrate in the presence of the first solution is smaller than acorresponding cross-sectional area of the second solution that would begenerated by dispensing the second solution onto the surface of thesubstrate in the absence of the first solution. In some aspects, thecross-sectional area of the second solution on the substrate generatedby dispensing the second solution onto the surface of the substrate inthe presence of the first solution is about two-fold, about three-fold,about four-fold, or about five-fold, about 10-fold, about 20-fold, about30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold,about 80-fold, about 90-fold, or about 100-fold smaller than thecorresponding cross-sectional area of the second solution that would begenerated by dispensing the second solution onto the surface of thesubstrate in the absence of the first solution.

Curing

Any suitable technique or condition can be used to cure the firstsolution, the second solution, and/or the spot formed by the secondsolution after the second solution (including the capture probes) isdispensed onto the substrate. As used herein, the term “curing” andrelated terms can refer to treating a solution (e.g., a first solution,a second solution, or both) with an agent and/or condition thattransforms a solution from a liquid state to a solid state (e.g.,transformed into a matrix), wherein the solution retains a threedimensional shape after the curing process. Suitable examples of curingtechniques and/or conditions include ultraviolet (UV) radiation,infrared (IR) radiation, thermal radiation, microwave radiation, visibleradiation, narrow-wavelength radiation, laser light, natural light,humidity, or combinations thereof. Suitable examples of a curing sourceinclude, for example, a UV light, a heating device, a radiation device,a microwave device, a plasma device, or combinations thereof.

In some embodiments, the first solution and/or the second solution ischemically cured. In some embodiments, the oligonucleotide and/orfeature droplet(s) are chemically cured. In some embodiments, the bulksolution is chemically cured. In some aspects, the spot formed by thesecond solution after the second solution (including the capture probes)is dispensed onto the substrate is chemically cured. In someembodiments, the oligonucleotides and/or features and the bulk solutionare attached to the substrate (e.g., by curing), thus generating afeature and bulk solution-matrix. In some aspects, the matrix (e.g., thefirst and second solution-matrix) is chemically cured. Chemically curinga solution can be accomplished by any means known in the art. Forexample, a solution can include one or more hydrogel subunits that canbe chemically polymerized (e.g., cross-linked) to form athree-dimensional (3D) hydrogel network. Features dispensed, in the formof a liquid droplet, onto the surface of a substrate in the presence ofa bulk solution can be polymerized. In some embodiments, the featuresare co-polymerized with the bulk solution to create a gel feature inhydrogel flexible array, whereas in other embodiments the gel pad orfeature is polymerized and the bulk solution is removed leaving a spotor bead array. Non-limiting examples of hydrogel subunits includeacrylamide, bis-acrylamide, polyacrylamide and derivatives thereof,poly(ethylene glycol) and derivatives thereof (e.g., PEG-acrylate(PEG-DA), PEG-RGD), gelatin-methacryloyl (GelMA), methacrylatedhyaluronic acid (MeHA), polyaliphatic polyurethanes, polyetherpolyurethanes, polyester polyurethanes, polyethylene copolymers,polyamides, polyvinyl alcohols, polypropylene glycol, polytetramethyleneoxide, polyvinyl pyrrolidone, polyacrylamide, poly(hydroxyethylacrylate), and poly(hydroxyethyl methacrylate), collagen, hyaluronicacid, chitosan, dextran, agarose, gelatin, alginate, protein polymers,methylcellulose, and the like, or combinations thereof. Other materialsand techniques useful in forming and/or cross-linking hydrogels aredescribed in more detail herein.

In some aspects, the first solution and/or second solution isphoto-reactively cured. In some embodiments, the oligonucleotide and/orfeature droplet(s) are photo-reactively cured. In some embodiments, thebulk solution is photo-reactively cured. In some aspects, the spotformed by the second solution after the second solution including thecapture probe is dispensed onto the substrate is photo-reactively cured.In some aspects, the matrix (e.g., the first and second solution-matrix)is photo-reactively cured. Photo-reactive curing can be accomplished byany means known in the art.

In some aspects, methods disclosed herein include curing the first(e.g., bulk) and/or second solution. In some aspects, methods disclosedherein include curing the spot formed by the second solution after thesecond solution (e.g., a second solution including the capture probe) isdispensed onto the substrate. In some aspects, methods disclosed hereininclude curing the first solution and the second solution after both aredispensed onto the slide. In some embodiments, curing the first andsecond solutions results in a first-and-second solution matrix. In someaspects, methods disclosed herein include curing the second solutionfollowed by removal of the uncured first solution from the substrate. Insome aspects, the methods disclosed herein include curing the first andsecond solutions on the substrate, thus generating a matrix (e.g., afirst and second solution-matrix).

Expanding Matrices

In some embodiments, prior to dispensing the second solution droplet(s)(e.g., oligonucleotide and/or feature droplet(s)) onto the surface of asubstrate in the presence of a first (e.g., bulk) solution, the first(e.g., bulk) solution is cured to form a bulk solution-matrix, and thebulk solution matrix is reversibly expanded along one or more axes(e.g., one or more axes of a matrix or a substrate surface). In someembodiments, second solution droplet(s) can be dispensed in the presenceof expanded first (e.g., bulk) solution-matrix.

In some embodiments, dispensing a plurality of second solutiondroplet(s) (e.g., including different capture probes having differentspatial barcodes) onto a substrate covered with a first (e.g., bulk)solution, wherein the first solution is initially stretched along one ormore axes. In some embodiments, the stretched first solution is cured.In some embodiments, the stretched first solution is partially cured. Insome embodiments, the stretched first solution is uncured and isdispensed on a surface that itself is stretched. In some aspects, thevolume (e.g., cross-sectional area) of the second solution droplet(s) isdecreased after being dispensed into a first solution that is initiallystretched. In some aspects, disclosed herein are methods of preparing anarray including (i) providing a gel or polymer (e.g., a cured orpartially cured solution) onto a substrate, (2) stretching the gel orpolymer, (3) dispensing a droplet of the second solution onto thesubstrate while the gel or polymer is stretched, and (4) allowing thegel or polymer to relax, thereby decreasing the overall area (e.g.,cross-sectional area) of the second solution droplet(s) on thesubstrate. In some aspects, the stretching step includes reversiblyexpanding the gel or polymer along one or more axes coplanar with thesurface of the substrate.

Removing Solution

In some embodiments, the first (e.g., bulk) solution can be removed fromthe substrate after the oligonucleotide and/or feature droplet(s) areattached to the substrate. In some aspects, the first (e.g., bulk)solution is removed after the second solution including the one or morecapture probes (e.g., plurality of second solutions that includedifferent capture probes) is dispensed onto the substrate. Methods ofremoving the first (e.g., bulk) solution are known in the art. In someaspects, removal of the first (e.g., bulk) solution results in completeremoval of the first solution, leaving only the second solutionincluding the one or more capture probes (e.g., plurality of secondsolutions that include different capture probes) on the substrate. Insome aspects, removal of the first (e.g., bulk) solution does not changethe surface area of the second solution (e.g., plurality of secondsolutions that include different capture probes) in contact with thesubstrate. In some aspects, removal of the first (e.g., bulk) solutiondoes not change the shape of the droplet of the second solution (e.g.,plurality of second solutions that include different capture probes) incontact with the substrate. In some aspects, prior to removal of thefirst (e.g., bulk) solution, the second solution including the one ormore probes (e.g., plurality of second solutions that include differentcapture probes) is cured by methods disclosed herein but that the first(e.g., bulk) solution is not cured. For example, the first and secondsolutions can be subjected to agents and/or conditions under which thesecond solution (e.g., plurality of second solutions that includedifferent capture probes) is cured, while the first (e.g., bulk)solution is not cured. In some embodiments, the second solution (e.g.,plurality of second solutions that include different capture probes)includes one or more hydrogel subunits that can be polymerized (e.g.,cross-linked) to form a three-dimensional (3D) hydrogel network, whilethe first (e.g., bulk) solution does not include such one or morehydrogel subunits. Upon subjecting the first and second solutions tocuring conditions, only the second solution(s) will be cured, allowingthe first solution to be removed.

In some embodiments, the first (e.g., bulk) solution is not removed fromthe substrate after the oligonucleotides and/or features are attached tothe substrate.

Shrinking Droplet / Feature Arrays

In some embodiments, an expanded first (e.g., bulk) solution-matrixcontaining a second solution (e.g., oligonucleotide and/or featuredroplet(s)) can be shrunk along one or more axes (e.g., of the matrix orof the substrate surface) such that the cross-sectional area of thesecond solution droplet(s) (e.g., oligonucleotide and/or featuredroplet(s)) is smaller than a corresponding cross-sectional area of asecond solution droplet(s) e.g., oligonucleotide and/or featuredroplet(s)) that would be generated if the first (e.g., bulk)solution-matrix containing the second solution droplet(s) (e.g.,oligonucleotide and/or feature droplet(s)) were not shrunk along the oneor more axes. In some embodiments, shrinking the matrix (e.g., thefirst-and-second solution matrix) generates a shrunken matrix (e.g., ashrunken first-and-second solution matrix), wherein the volume of asecond solution droplet (e.g., a plurality of second solution droplets)of the matrix is reduced as compared to the volume of the secondsolution droplet in a non-shrunken matrix. In some aspects, shrinkingtwo or more second solutions (e.g., droplets of second solutions)results in a higher density of the two or more second solutions. In someembodiments, the second solution droplet(s) (e.g., oligonucleotideand/or feature droplet(s)) and first (e.g., bulk) solution-matrix can beshrunk by any method disclosed herein. In some embodiments, theshrinking can include removing water. In some aspects, the resolution ofthe array is increased after the droplet is shrunk. In some aspects,shrinking the second solution droplet results in a decrease in thecross-sectional area of the droplet (e.g., the cross-sectional area inthe plane of the substrate onto which the second solution droplet isprinted or dispensed). In some aspects, after shrinking the droplet, theconcentration of probes on the substrate will be increased, therebyimproving sensitivity.

The spatial array is contacted to the biological sample, wherein thebiological sample can be any described herein (e.g., a FFPE tissuesection). Once the spatial array has been placed on the biologicalsample, a cellular and/or nuclear permeabilization reaction can occur,such that the biological analytes (e.g., DNA, RNA, proteins,metabolites, small molecules, lipids, and the like) are released andcaptured onto the spatial array, preserving their spatial information.The spatial array is removed, and the molecular information therein isdetermined (e.g., by performing library construction for next generationsequencing). Sequencing can be followed by correlation of the expressionvalue (e.g., gene expression of the analyte) with the feature.

In some aspects, the cross-sectional area of a second solution dropletis decreased upon shrinking by about 10%, by about 20%, by about 30%, byabout 40%, by about 50%, by about 60%, by about 70%, by about 80%, or byabout 90% compared to the cross-sectional area of a second solutiondroplet that is printed or dispensed onto the substrate. In someaspects, the cross-sectional area of a second solution droplet isdecreased by about 1.0-fold, by about 1.1-fold, by about 1.2-fold, byabout 1.3-fold, by about 1.4 fold, by about 1.5-fold, by about 2-fold,by about 3-fold, by about 4-fold, or by about 5-fold compared to thecross-sectional area of a second solution droplet that is printed ordispensed onto the substrate.

In some aspects, disclosed herein are methods of preparing an arrayincluding dispensing a plurality of second solution droplets of (e.g.,including different capture probes having different spatial barcodes)onto the substrate covered with a first (e.g., bulk) solution, curingthe first and second solutions to generate a matrix (e.g., a first andsecond solution-matrix), wherein the volume and shape of the pluralityof second solution droplets in the matrix are substantially the same aswhen the plurality of second solution droplets was dispensed, andshrinking the matrix, thereby decreasing the overall volume (e.g.,cross-sectional area) of the plurality of second solution droplets. Insome aspects, shrinking can be accomplished using any of the variety ofshrinking agents or conditions described herein. In some aspects,shrinking can be accomplished by dehydrating the matrix.

In some aspects, after the second solution is dispensed, the area of thesecond solution is shrunk along the one or more axes of coplanar withthe surface of the substrate such that the cross-sectional area of thesecond solution on the substrate (e.g., the slide) is smaller than acorresponding cross-sectional area of the second solution that would begenerated if the first solution matrix including the second solutionwere not shrunk along the one or more axes of the surface of thesubstrate.

With respect to orientation, shrinkage need not be equal in any twoorthogonal directions on the substrate. However, in some aspects, theshrinkage of a second solution droplet is substantially uniform inshrinkage. In some aspects, a second solution droplet shrinks insubstantially the same amount in each direction, regardless of positionon the substrate plane.

In some aspects, after preparing the substrate with the one or morecapture probes (e.g., a spatial array that includes the capture probes),disclosed herein are methods for spatially profiling an analyte (e.g., aplurality of analytes) in a biological sample, including (a) generatinga spatial array including a plurality of capture probes bound to asubstrate; wherein (i) at least a portion of the substrate is coatedwith a first solution; and (ii) a plurality of second solutions, in theform of a liquid droplets, are dispensed onto the surface of thesubstrate in the presence of the first solution, wherein the firstsolution and the plurality of second solutions do not substantially mixwith each other, wherein at least two members of the plurality of thesecond solutions include one or more different capture probes havingdifferent spatial barcodes, and wherein at least one of the one or morecapture probes of at least two members of the plurality of secondsolutions is bound to the substrate; (b) contacting the biologicalsample with the spatial array such that the analyte(s) present in thebiological sample are captured by one or more of the capture probes; and(c) determining the spatial profile of the captured analyte(s) in thebiological sample.

(XII) Array / Feature Preservation

In some embodiments, the biological sample can be preserved aftercompletion of an assay with a feature or arrangement of features foradditional rounds of spatial detection of analytes. In some embodiments,the biological sample, features, array, or any combination thereof canbe preserved after the spatial profiling. In some embodiments, thebiological sample, features, array, or combinations thereof can beprotected from dehydration (e.g., drying, desiccation). In someembodiments, the biological sample, features, array, or combinationsthereof, can be protected from evaporation. Methods of preserving and/orprotecting biological samples, features, or arrays are known in the art.For example, in a non-limiting way, the biological sample, features,array, or combinations thereof can be covered by a reversible sealingagent. Any suitable reversible sealing agent can be used. Methods ofreversible sealing are known in the art (See, e.g., WO 2019/104337,which is incorporated herein by reference). In a non-limiting way,suitable reversible sealing agents can include non-porous materials,membranes, lids, or oils (e.g., silicone oil, mineral oil). In furthernon-limiting examples, the biological sample, features, array, orcombinations thereof can be preserved in an environmental chamber (e.g.,hermetically sealed) and removed for additional rounds of spatialanalysis at a later time.

(E) Analyte Capture

In this section, general aspects of methods and systems for capturinganalytes are described. Individual method steps and system features canbe present in combination in many different embodiments; the specificcombinations described herein do not in any way limit other combinationsof steps or features.

(I) Conditions for Capture

Generally, analytes can be captured when contacting a biological samplewith a substrate including capture probes (e.g., substrate with captureprobes embedded, spotted, printed on the substrate or a substrate withfeatures (e.g., beads, wells) comprising capture probes).

As used herein, “contact,” “contacted,” and/ or “contacting,” abiological sample with a substrate refers to any contact (e.g., director indirect) such that capture probes can interact (e.g., capture) withanalytes from the biological sample. For example, a substrate may benear or adjacent to the biological sample without direct physicalcontact, yet capable of capturing analytes from the biological sample.In some embodiments a biological sample is in direct physical contactwith a substrate. In some embodiments, a biological sample is inindirect physical contact with a substrate. For example, a liquid layermay be between the biological sample and the substrate. In someembodiments, analytes diffuse through a liquid layer. In someembodiments capture probes diffuse through a liquid layer. In someembodiments reagents may be delivered via a liquid layer between abiological sample and a substrate. In some embodiments, indirectphysical contact may include a second substrate (e.g., a hydrogel, afilm, a porous membrane) between the biological sample and the firstsubstrate comprising capture probes. In some embodiments, reagents maybe delivered by a second substrate to a biological sample.

In some embodiments, a cell immobilization agent can be used to contacta biological sample with a substrate (e.g., by immobilizingnon-aggregated or disaggregated sample on a spatially-barcoded arrayprior to analyte capture). A “cell immobilization agent” as used hereincan refer to an agent (e.g., an antibody), attached to a substrate,which can bind to a cell surface marker. Non-limiting examples of a cellsurface marker include CD45, CD3, CD4, CD8, CD56, CD19, CD20, CD11c,CD14, CD33, CD66b, CD34, CD41, CD61, CD235a, CD146, and epithelialcellular adhesion molecule (EpCAM). A cell immobilization agent caninclude any probe or component that can bind to (e.g., immobilize) acell or tissue when on a substrate. A cell immobilization agent attachedto the surface of a substrate can be used to bind a cell that has a cellsurface maker. The cell surface marker can be a ubiquitous cell surfacemarker, wherein the purpose of the cell immobilization agent is tocapture a high percentage of cells within the sample. The cell surfacemarker can be a specific, or more rarely expressed, cell surface marker,wherein the purpose of the cell immobilization agent is to capture aspecific cell population expressing the target cell surface marker.Accordingly, a cell immobilization agent can be used to selectivelycapture a cell expressing the target cell surface marker from apopulation of cells that do not have the same cell surface marker.

Capture probes on a substrate (or on a feature on the substrate) mayinteract with released analytes through a capture domain, describedelsewhere, to capture analytes. In some embodiments, certain steps areperformed to enhance the transfer or capture of analytes to the captureprobes of the array. Examples of such modifications include, but are notlimited to, adjusting conditions for contacting the substrate with abiological sample (e.g., time, temperature, orientation, pH levels,pre-treating of biological samples, etc.), using force to transportanalytes (e.g., electrophoretic, centrifugal, mechanical, etc.),performing amplification reactions to increase the amount of biologicalanalytes (e.g., PCR amplification, in situ amplification, clonalamplification), and/or using labeled probes for detecting of ampliconsand barcodes.

In some embodiments, an array is adapted in order to facilitatebiological analyte migration. Non-limiting examples of adapting an arrayto facilitate biological analyte migration include arrays withsubstrates containing nanopores, nanowells, and/or microfluidicchannels; arrays with porous membranes; and arrays with substrates thatare made of hydrogel. In some cases, the array substrate is liquidpermeable. In some cases, the array is a coverslip or slide thatincludes nanowells or patterning, (e.g., via fabrication). In some caseswhere the substrate includes nanopores, nanowells, and/or microfluidicchannels, these structures can facilitate exposure of the biologicalsample to reagents (e.g., reagents for permeabilization, biologicalanalyte capture, and/or a nucleic acid extension reaction), therebyincreasing analyte capture efficiency as compared to a substrate lackingsuch characteristics.

In some embodiments, analyte capture is facilitated by treating abiological sample with permeabilization reagents. If a biological sampleis not permeabilized sufficiently, the amount of analyte captured on asubstrate can be too low to enable adequate analysis. Conversely, if abiological sample is too permeable, an analyte can diffuse away from itsorigin in the biological sample, such that the relative spatialrelationship of the analytes within the biological sample is lost.Hence, a balance between permeabilizing the biological sample enough toobtain good signal intensity while still maintaining the spatialresolution of the analyte distribution in the biological sample isdesired. Methods of preparing biological samples to facilitate analytecapture are known in the art and can be modified depending on thebiological sample and how the biological sample is prepared (e.g., freshfrozen, FFPE, etc.).

(II) Substrate Holder

Described herein are methods in which an array with capture probeslocated on a substrate and a biological sample located on a differentsubstrate, are contacted such that the array is in contact with thebiological sample (e.g., the substrates are sandwiched together). Insome embodiments, the array and the biological sample can be contacted(e.g., sandwiched), without the aid of a substrate holder. In someembodiments, the array and biological sample substrates can be placed ina substrate holder (e.g., an array alignment device) designed to alignthe biological sample and the array. For example, the substrate holdercan have placeholders for two substrates. In some embodiments, an arrayincluding capture probes can be positioned on one side of the substrateholder (e.g., in a first substrate placeholder). In some embodiments, abiological sample can be placed on the adjacent side of the substrateholder in a second placeholder. In some embodiments, a hinge can belocated between the two substrate placeholders that allows the substrateholder to close, e.g., make a sandwich between the two substrateplaceholders. In some embodiments, when the substrate holder is closedthe biological sample and the array with capture probes are contactedwith one another under conditions sufficient to allow analytes presentin the biological sample to interact with the capture probes of thearray. For example, dried permeabilization reagents can be placed on thebiological sample and rehydrated. A permeabilization solution can beflowed through the substrate holder to permeabilize the biologicalsample and allow analytes in the biological sample to interact with thecapture probes. Additionally, the temperature of the substrates orpermeabilization solution can be used to initiate or control the rate ofpermeabilization. For example, the substrate including the array, thesubstrate including the biological sample, or both substrates can beheld at a low temperature to slow diffusion and permeabilizationefficiency. Once sandwiched, in some embodiments, the substrates can beheated to initiate permeabilization and/or increase diffusionefficiency. Transcripts that are released from the permeabilized tissuecan diffuse to the array and be captured by the capture probes. Thesandwich can be opened, and cDNA synthesis can be performed on thearray.

Any of the variety of combinations described herein where a sandwichincluding an array with capture probes and a biological sample on twodifferent substrates can be placed in a substrate holder designed toalign the biological sample and the array. For example, the substrateholder can have placeholders for two substrates. In some embodiments, anarray including capture probes can be positioned on one side of thesubstrate holder (e.g., in a first substrate placeholder). In someembodiments, a biological sample can be placed on the adjacent side ofthe substrate holder in a second placeholder. In some embodiments, inbetween the two substrate placeholders can be a hinge that allows thesubstrate holder to close, e.g., make a sandwich between the twosubstrate placeholders. In some embodiments, when the substrate holderis closed the biological sample and the array with capture probes can becontacted with one another under conditions sufficient to allow analytespresent in the biological sample to interact with the capture probes ofthe array for spatial analysis by any method described herein. Forexample, dried permeabilization reagents can be placed on the biologicalsample and rehydrated. Additionally, a permeabilization solution can beflowed through the substrate holder to permeabilize the biologicalsample and allow analytes in the biological sample to interact with thecapture probes.

In some embodiments, a flexible array described herein can be placed inthe substrate holder, and sandwiched with a biological sample. In someembodiments, the flexible array can include spatially-barcodedcross-linked features. In some embodiments, the flexible array can bepresoaked in permeabilization reagents before being placed into thesubstrate holder. In some embodiments, the flexible array can be soakedin permeabilization reagents after being placed in the substrate holder.In some embodiments, the substrate holder including a biological samplein one placeholder and a flexible array can be closed (e.g., form asandwich) such that the permeabilization reagents allow analytes presentin the biological sample to interact with capture probes of the flexiblearray (e.g., capture probes on the spatially-barcoded features).

In some embodiments, the substrate holder can be heated or cooled toregulate permeabilization and/or diffusion efficiency.

(III) Passive Capture Methods

In some embodiments, analytes can be migrated from a sample to asubstrate. Methods for facilitating migration can be passive (e.g.,diffusion) and/or active (e.g., electrophoretic migration of nucleicacids). Non-limiting examples of passive migration can include simplediffusion and osmotic pressure created by the rehydration of dehydratedobjects.

Passive migration by diffusion uses concentration gradients. Diffusionis movement of untethered objects toward equilibrium. Therefore, whenthere is a region of high object concentration and a region of lowobject concentration, the object (e.g., a capture probe, an analyte,etc.) moves to an area of lower concentration. In some embodiments,untethered analytes move down a concentration gradient.

In some embodiments, different reagents may be added to the biologicalsample, such that the biological sample is rehydrated while improvingcapture of analytes. In some embodiments, the biological sample can becontacted with a shrunken array as described herein. In someembodiments, the biological sample and/or the shrunken array can berehydrated with permeabilization reagents. In some embodiments, thebiological sample and/or the shrunken array can be rehydrated with astaining solution (e.g., hematoxylin and eosin stain).

(IV) Diffusion-Resistant Media / Lids

To increase efficiency by encouraging analyte diffusion toward thespatially-barcoded capture probes, a diffusion-resistant medium can beused. In general, molecular diffusion of biological analytes occurs inall directions, including toward the capture probes (i.e., toward thespatially-barcoded array), and away from the capture probes (i.e., intothe bulk solution). Increasing diffusion toward the spatially-barcodedarray reduces analyte diffusion away from the spatially-barcoded arrayand increases the capturing efficiency of the capture probes.

In some embodiments, a biological sample is placed on the top of aspatially-barcoded substrate and a diffusion-resistant medium is placedon top of the biological sample. For example, the diffusion-resistantmedium can be placed onto an array that has been placed in contact witha biological sample. In some embodiments, the diffusion-resistant mediumand spatially-barcoded array are the same component. For example, thediffusion-resistant medium can contain spatially-barcoded capture probeswithin or on the diffusion-resistant medium (e.g., coverslip, slide,hydrogel, or membrane). In some embodiments, a sample is placed on asubstrate and a diffusion-resistant medium is placed on top of thebiological sample. Additionally, a spatially-barcoded capture probearray can be placed in close proximity over a diffusion-resistantmedium. For example, a diffusion-resistant medium may be sandwichedbetween a spatially-barcoded array and a sample on a substrate. In someembodiments, a diffusion-resistant medium is disposed or spotted onto asample. In other embodiments, a diffusion-resistant medium is placed inclose proximity to a sample.

In general, a diffusion-resistant medium can be any material known tolimit diffusivity of biological analytes. For example, adiffusion-resistant medium can be a solid lid (e.g., coverslip or glassslide). In some embodiments, a diffusion-resistant medium may be made ofglass, silicon, paper, hydrogel polymer monoliths, or other material. Insome embodiments, the glass side can be an acrylated glass slide. Insome embodiments, the diffusion-resistant medium is a porous membrane.In some embodiments, the material may be naturally porous. In someembodiments, the material may have pores or wells etched into solidmaterial. In some embodiments, the pore volume can be manipulated tominimize loss of target analytes. In some embodiments, the membranechemistry can be manipulated to minimize loss of target analytes. Insome embodiments, the diffusion-resistant medium (e.g., hydrogel) iscovalently attached to a substrate (e.g., glass slide). In someembodiments, a diffusion-resistant medium can be any material known tolimit diffusivity of poly(A) transcripts. In some embodiments, adiffusion-resistant medium can be any material known to limit thediffusivity of proteins. In some embodiments, a diffusion-resistantmedium can be any material know to limit the diffusivity ofmacromolecular constituents.

In some embodiments, a diffusion-resistant medium includes one or morediffusion-resistant media. For example, one or more diffusion-resistantmedia can be combined in a variety of ways prior to placing the media incontact with a biological sample including, without limitation, coating,layering, or spotting. As another example, a hydrogel can be placed ontoa biological sample followed by placement of a lid (e.g., glass slide)on top of the hydrogel.

In some embodiments, a force (e.g., hydrodynamic pressure, ultrasonicvibration, solute contrasts, microwave radiation, vascular circulation,or other electrical, mechanical, magnetic, centrifugal, and/or thermalforces) is applied to control diffusion and enhance analyte capture. Insome embodiments, one or more forces and one or more diffusion-resistantmedia are used to control diffusion and enhance capture. For example, acentrifugal force and a glass slide can used contemporaneously. Any of avariety of combinations of a force and a diffusion-resistant medium canbe used to control or mitigate diffusion and enhance analyte capture.

In some embodiments, a diffusion-resistant medium, along with thespatially-barcoded array and sample, is submerged in a bulk solution. Insome embodiments, a bulk solution includes permeabilization reagents. Insome embodiments, a diffusion-resistant medium includes at least onepermeabilization reagent. In some embodiments, a diffusion-resistantmedium (i.e. hydrogel) is soaked in permeabilization reagents beforecontacting the diffusion-resistant medium to the sample. In someembodiments, a diffusion-resistant medium can include wells (e.g.,micro-, nano-, or picowells) containing a permeabilization buffer orreagents. In some embodiments, a diffusion-resistant medium can includepermeabilization reagents. In some embodiments, a diffusion-resistantmedium can contain dried reagents or monomers to deliverpermeabilization reagents when the diffusion-resistant medium is appliedto a biological sample. In some embodiments, a diffusion-resistantmedium is added to the spatially-barcoded array and sample assemblybefore the assembly is submerged in a bulk solution. In someembodiments, a diffusion-resistant medium is added to thespatially-barcoded array and sample assembly after the sample has beenexposed to permeabilization reagents. In some embodiments,permeabilization reagents are flowed through a microfluidic chamber orchannel over the diffusion-resistant medium. In some embodiments, theflow controls the sample’s access to the permeabilization reagents. Insome embodiments, target analytes diffuse out of the sample and toward abulk solution and get embedded in a spatially-barcoded captureprobe-embedded diffusion-resistant medium. In some embodiments, a freesolution is sandwiched between the biological sample and adiffusion-resistant medium.

FIG. 13 is an illustration of an exemplary use of a diffusion-resistantmedium. A diffusion-resistant medium 1302 can be contacted with a sample1303. In FIG. 13 , a glass slide 1304 is populated withspatially-barcoded capture probes 1306, and the sample 1303, 1305 iscontacted with the array 1304, 1306. A diffusion-resistant medium 1302can be applied to the sample 1303, wherein the sample 1303 is sandwichedbetween a diffusion-resistant medium 1302 and a capture probe coatedslide 1304. When a permeabilization solution 1301 is applied to thesample, using the diffusion-resistant medium/lid 1302 directs migrationof the analytes 1305 toward the capture probes 1306 by reducingdiffusion of the analytes out into the medium. Alternatively, the lidmay contain permeabilization reagents.

(V) Active Capture Methods

In some of the methods described herein, an analyte in a biologicalsample (e.g., in a cell or tissue section) can be transported (e.g.,passively or actively) to a capture probe (e.g., a capture probe affixedto a substrate (e.g., a substrate or bead)).

For example, analytes can be transported to a capture probe (e.g., animmobilized capture probe) using an electric field (e.g., usingelectrophoresis), pressure, fluid flow, gravity, temperature, and/or amagnetic field. For example, analytes can be transported through, e.g.,a gel (e.g., hydrogel), a fluid, or a permeabilized cell, to a captureprobe (e.g., an immobilized capture probe) using a pressure gradient, achemical concentration gradient, a temperature gradient, and/or a pHgradient. For example, analytes can be transported through a gel (e.g.,hydrogel), a fluid, or a permeabilized cell, to a capture probe (e.g.,an immobilized capture probe).

In some examples, an electrophoretic field can be applied to analytes tofacilitate migration of analytes towards a capture probe. In someexamples, a sample containing analytes contacts a substrate havingcapture probes fixed on the substrate (e.g., a slide, cover slip, orbead), and an electric current is applied to promote the directionalmigration of charged analytes towards capture probes on a substrate. Anelectrophoresis assembly (e.g., an electrophoretic chamber), where abiological sample is in contact with a cathode and capture probes (e.g.,capture probes fixed on a substrate), and where the capture probes arein contact with the biological sample and an anode, can be used to applythe current.

In some embodiments, methods utilizing an active capture method canemploy a conductive substrate (e.g., any of the conductive substratesdescribed herein). In some embodiments, a conductive substrate includespaper, a hydrogel film, or a glass slide having a conductive coating. Insome embodiments, a conductive substrate (e.g., any of the conductivesubstrates described herein) includes one or more capture probes.

In some embodiments, electrophoretic transfer of analytes can beperformed while retaining the relative spatial locations of analytes ina biological sample while minimizing passive diffusion of an analyteaway from its location in a biological sample. In some embodiments, ananalyte captured by a capture probe (e.g., capture probes on asubstrate) retains the spatial location of the analyte present in thebiological sample from which it was obtained (e.g., the spatial locationof the analyte that is captured by a capture probe on a substrate whenthe analyte is actively migrated to the capture probe by electrophoretictransfer can be more precise or representative of the spatial locationof the analyte in the biological sample than when the analyte is notactively migrated to the capture probe). In some embodiments,electrophoretic transport and binding process is described by theDamköhler number (Da), which is a ratio of reaction and mass transportrates. The fraction of analytes bound and the shape of the biologicalsample will depend on the parameters in the Da. There parameters includeelectromigration velocity U_(e)(depending on analyte electrophoreticmobility µ_(e) and electric field strength E), density of capture probes(e.g., barcoded oligonucleotides) p₀, the binding rate between probes(e.g., barcoded oligonucleotides) and analytes k_(on), and capture areathickness L.

$\text{Da} \sim \frac{k_{on}p_{0}L}{\text{μ}_{e}E}$

Fast migration (e.g., electromigration) can reduce assay time and canminimize molecular diffusion of analytes.

In some embodiments, electrophoretic transfer of analytes can beperformed while retaining the relative spatial alignment of the analytesin the sample. As such, an analyte captured by the capture probes (e.g.,capture probes on a substrate) retains the spatial information of thecell or the biological sample from which it was obtained. Applying anelectrophoretic field to analytes can also result in an increase intemperature (e.g., heat). In some embodiments, the increased temperature(e.g., heat) can facilitate the migration of the analytes towards acapture probe.

In some examples, a spatially-addressable microelectrode array is usedfor spatially-constrained capture of at least one charged analyte ofinterest by a capture probe. For example, a spatially-addressablemicroelectrode array can allow for discrete (e.g., localized)application of an electric field rather than a uniform electric field.The spatially-addressable microelectrode array can be independentlyaddressable. In some embodiments, the electric field can be applied toone or more regions of interest in a biological sample. The electrodesmay be adjacent to each other or distant from each other. Themicroelectrode array can be configured to include a high density ofdiscrete sites having a small area for applying an electric field topromote the migration of charged analyte(s) of interest. For example,electrophoretic capture can be performed on a region of interest using aspatially-addressable microelectrode array.

A high density of discrete sites on a microelectrode array can be used.The surface can include any suitable density of discrete sites (e.g., adensity suitable for processing the sample on the conductive substratein a given amount of time). In one embodiment, the surface has a densityof discrete sites greater than or equal to about 500 sites per 1 mm². Insome embodiments, the surface has a density of discrete sites of about100, about 200, about 300, about 400, about 500, about 600, about 700,about 800, about 900, about 1,000, about 2,000, about 3,000, about4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000,about 10,000, about 20,000, about 40,000, about 60,000, about 80,000,about 100,000, or about 500,000 sites per 1 mm². In some embodiments,the surface has a density of discrete sites of at least about 200, atleast about 300, at least about 400, at least about 500, at least about600, at least about 700, at least about 800, at least about 900, atleast about 1,000, at least about 2,000, at least about 3,000, at leastabout 4,000, at least about 5,000, at least about 6,000, at least about7,000, at least about 8,000, at least about 9,000, at least about10,000, at least about 20,000, at least about 40,000, at least about60,000, at least about 80,000, at least about 100,000, or at least about500,000 sites per 1 mm².

Schematics illustrating an electrophoretic transfer system configured todirect nucleic acid analytes (e.g., mRNA transcripts) toward aspatially-barcoded capture probe array are shown in FIG. 14A and FIG.14B. In this exemplary configuration of an electrophoretic system, asample 1402 is sandwiched between the cathode 1401 and thespatially-barcoded capture probe array 1404, 1405, and thespatially-barcoded capture probe array 1404, 1405 is sandwiched betweenthe sample 1402 and the anode 1403, such that the sample 1402, 1406 isin contact with the spatially-barcoded capture probes 1407. When anelectric field is applied to the electrophoretic transfer system,negatively charged nucleic acid analytes 1406 will be pulled toward thepositively charged anode 1403 and into the spatially-barcoded array1404, 1405 containing the spatially-barcoded capture probes 1407. Thespatially-barcoded capture probes 1407 interact with the nucleic acidanalytes (e.g., mRNA transcripts hybridize to spatially-barcoded nucleicacid capture probes forming DNA/RNA hybrids) 1406, making the analytecapture more efficient. The electrophoretic system set-up may changedepending on the target analyte. For example, proteins may be positive,negative, neutral, or polar depending on the protein as well as otherfactors (e.g., isoelectric point, solubility, etc.). The skilledpractitioner has the knowledge and experience to arrange theelectrophoretic transfer system to facilitate capture of a particulartarget analyte.

FIG. 15 is an illustration showing an exemplary workflow protocolutilizing an electrophoretic transfer system. In the example, Panel Adepicts a flexible spatially-barcoded feature array being contacted witha sample. The sample can be a flexible array, wherein the array isimmobilized on a hydrogel, membrane, or other flexible substrate. PanelB depicts contact of the array with the sample and imaging of thearray-sample assembly. The image of the sample/array assembly can beused to verify sample placement, choose a region of interest, or anyother reason for imaging a sample on an array as described herein. PanelC depicts application of an electric field using an electrophoretictransfer system to aid in efficient capture of a target analyte. Here,negatively charged mRNA target analytes migrate toward the positivelycharged anode. Panel D depicts application of reverse transcriptionreagents and first strand cDNA synthesis of the captured targetanalytes. Panel E depicts array removal and preparation for libraryconstruction (Panel F) and next-generation sequencing (Panel G).

(VI) Targeted Analysis

In some aspects, arrays (e.g., glass slides) include a plurality ofcapture probes that bind to one or more specific biological targets in asample. The capture probes can be directly or indirectly attached to asubstrate. The capture probe can be or include, for example, DNA or RNA.In some aspects, the capture probes on an array can be immobilized,e.g., attached or bound, to the array via their 5′ or 3′ ends, dependingon the chemical matrix of the array. In some aspects, the probes areattached via a 3′ linkage, thereby leaving a free 5′ end. In someaspects, the probes are attached via a 5′ linkage, thereby leaving afree 3′ end. In some aspects, the probes are immobilized indirectly. Forexample, a probe can be attached to a bead, which bead can be depositedon a substrate. A capture probe as disclosed in this section can includeany of the various components of a capture probe as provided throughoutthis disclosure (e.g., spatial barcodes, UMIs, functional domains,cleavage domains, etc.).

In some aspects, a capture probe or plurality of capture probes interactwith an analyte specific for a particular species or organism (e.g.,host or pathogen). In some aspects, the probe or plurality of probes canbe used to detect a viral, bacterial, or plant protein or nucleic acid.In some aspects, the capture probe or plurality of capture probes can beused to detect the presence of a pathogen (e.g., bacteria or virus) inthe biological sample. In some aspects, the capture probe or pluralityof capture probes can be used to detect the expression of a particularnucleic acid associated with a pathogen (e.g., presence of 16S ribosomalRNA or Human Immunodeficiency Virus (HIV) RNA in a human sample).

In some aspects, the capture domain in the capture probe can interactwith one or more specific analytes (e.g., an analyte or a subset ofanalytes out of the pool of total analytes). The specific analyte(s) tobe detected can be any of a variety of biological molecules includingbut not limited to proteins, nucleic acids, lipids, carbohydrates, ions,small molecules, subcellular targets, or multicomponent complexescontaining any of the above. In some embodiments, the analyte(s) can belocalized to subcellular location(s), including, for example,organelles, e.g., mitochondria, Golgi apparatus, endoplasmic reticulum,chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles,lysosomes, etc. In some embodiments, analyte(s) can be peptides orproteins, including without limitation antibodies and enzymes.

In some aspects, analytes from a biological sample interact with one ormore capture probes (e.g., one or more capture probes immobilizeddirectly or indirectly on a substrate), and the capture probes interactwith specific analytes in the biological sample. In some aspects, thecapture probes are allowed to interact with (e.g., hybridize to)specific analytes, e.g., under appropriate conditions whereoligonucleotide capture probes can hybridize to the target nucleicacids. In some aspects, analytes that did not hybridize to captureprobes are removed (e.g., analytes that do not interact with capturedomains of the capture probes). In some embodiments, removal of analytesthat did not interact with a capture probe can be accomplished by, e.g.,washing the sample to remove such analytes.

In some aspects, a capture probe or plurality of capture probes includesa capture domain that interacts with an analyte or analytes present in abiological sample. In some aspects, the capture probe or plurality ofcapture probes includes a capture domain that detects the presence orlevel amount (e.g., expression level) of a particular analyte oranalytes of interest. The capture domain of a capture probe (immobilizeddirectly or indirectly on a substrate) can be capable of bindingselectively to a desired subtype or subset of nucleic acid. In someaspects, for example, the capture domain binds to a subset of nucleicacids in a genome or a subset of nucleic acids in a transcriptome. Insome aspects, the analyte(s) can include one or more nucleic acids. Insome aspects, the capture probe or plurality of capture probes can beused to detect the expression of a particular transcript (e.g., aparticular mRNA). In some aspects, a capture probe or plurality ofcapture probes can be specific for (e.g., binds to) an individual changein a nucleic acid or protein (e.g., a mutation or single nucleotidepolymorphism (SNP)).

In some aspects, the biological sample includes an analyte that is orincludes a nucleic acid. The nucleic acid can be RNA or DNA. In someaspects, the capture probe or plurality of capture probes detects DNAcopy number of a particular set of nucleic acid analyte or analytes. Forexample, capture probe or plurality of capture probes provided hereincan be used to detect DNA copy number of nucleic acids that sharehomology to each other.

In some aspects, the capture probe or plurality of capture probesincludes a capture domain that detects the presence or level amount(e.g., expression level) of one or more RNA transcripts (e.g., specificRNA transcripts). In some aspects, the capture probe or plurality ofcapture probes includes a capture domain that detects the presence oramount (e.g., expression level) of one or more non-coding RNAs (e.g.,microRNA, transfer RNA (tRNA), ribosomal RNA (rRNA), small interferingRNA (siRNA) and small nucleolar RNA (snoRNA). In some aspects, the probeor plurality of probes includes a capture domain that detects thepresence or level amount (e.g., expression level) of one or moreproteins (e.g., proteins expressed of a nucleic acid of interest).

In some aspects, the capture probe or plurality of capture probes can bespecific for a particular protein. In some aspects, the capture probe orplurality of capture probes can be used to detect the presence of aparticular protein of interest. In some aspects, the capture probe orplurality of capture probes can be used to detect translation of aparticular protein. In some aspects, the capture probe or plurality ofcapture probes can specifically interact with an active region of anenzyme, a binding domain of an immunoglobulin, defined domains ofproteins, whole proteins, synthetic peptides, peptides with introducedmutations, aptamer, or any combination thereof. In some aspects, theanalyte(s) can include one or more proteins. In some aspects, theanalyte(s) can include one or more nucleic acids and one or moreproteins.

In some aspects, the capture probe or plurality of capture probes can beused to detect particular post-translational modifications of aparticular protein. In such embodiments, analyte capture agents can bespecific for cell surface analytes having a given state ofposttranslational modification (e.g., phosphorylation, glycosylation,ubiquitination, nitrosylation, methylation, acetylation or lipidation),such that a cell surface analyte profile can include posttranslationalmodification information of one or more analytes.

In some aspects, the capture probe or plurality of capture probes can bespecific for a particular set of nucleic acids (e.g., nucleic acids thatare associated with a specific cellular pathway or pathways). In someaspects, the set of nucleic acids is DNA. In some aspects, the set ofnucleic acids is RNA. In some aspects, the set of nucleic acids hassimilar and/or homologous sequences. In some aspects, the set of nucleicacids encodes for analytes that function in a similar cellular pathway.In some aspects, the set of nucleic acids encodes for analytes that areexpressed in a certain pathological state (e.g., cancer, Alzheimer’s, orParkinson’s disease). In some aspects, the set of nucleic acids encodesfor analytes that are over-expressed in a certain pathological state. Insome aspects, the set of nucleic acids encodes for analytes that areunder-expressed in a certain pathological state.

In some aspects, the capture probe or plurality of capture probes can bespecific for a particular nucleic acid, or detection or expression of aparticular set of proteins (e.g., in a similar cellular pathway). Insome aspects, the set of proteins has similar functional domains. Insome aspects, the set of proteins functions in a similar cellularpathway. In some aspects, the set of proteins is expressed in a certainpathological state (e.g., cancer, Alzheimer’s or Parkinson’s disease).In some aspects, the set of proteins is over-expressed in a certainpathological state. In some aspects, the set of proteins isunder-expressed in a certain pathological state.

In some embodiments, a capture probe includes a capture domain that iscapable of binding to more than one analyte. In some embodiments, acapture domain can bind to one or more analytes that are about 80%identical, about 85% identical, about 90% identical, about 95%identical, about 96% identical, about 97% identical, about 98%identical, about 99% identical, 100% identical to the target analyte. Insome aspects, the capture probe can bind to an analyte that is about 80%identical, about 85% identical, about 90% identical, about 95%identical, about 96% identical, about 97% identical, about 98%identical, or about 99% identical to each other. In some embodiments, acapture domain can bind to a conserved region of one or more analytes,in which the conserved regions are about 80% identical, about 85%identical, about 90% identical, about 95% identical, about 96%identical, about 97% identical, about 98% identical, about 99%identical, 100% identical to the target analyte.

In some aspects, a capture probe or plurality of capture probesinteracts with two or more analytes (e.g., nucleic acids or proteins)that are not similar in sequence and/or do not share a conserved domain.In some embodiments, a capture probe includes two or more capturedomains, each of which interacts with a different analyte. In suchembodiments, members of the two or more capture domains can be adjacentto each other in the capture probe and/or members of the two or morecapture domains can be separated from each other in the capture probe byone or more domains (e.g., nucleic acid domains). For example, in someaspects, the sets of analytes that are detected include mutationalchanges in the targeted nucleic acids or proteins. In some aspects, thecapture probe or plurality of capture probes detects sets of nucleicacids or proteins (e.g., non-homologous nucleic acids or proteins) thatare individually mutated during a pathogenic state. In some aspects, thepathogenic state is cancer.

In some aspects, a capture probe or plurality of capture probes includecapture domains that can be used to detect analytes that are typicallydetected using diagnostic panels. In some aspects, the capture probe orplurality of capture probes are used to detect changes in one or moreanalytes. In some aspects, the analyte changes include one or more ofincreased analyte expression, decreased analyte expression, mutatednucleic acid sequences, or any combination thereof. In some aspects, thechanges in the analytes are associated with and/or lead to manifestationof a pathogenic state in a subject. In some aspects, the detectedchanges are compared to a reference analyte or analytes.

(VII) Polypeptide Capture

Provided herein are methods and materials for identifying the locationof a polypeptide in a biological sample. In some embodiments, an analyte(e.g., a polypeptide analyte) can be directly captured on a substrate.For example, polypeptide analytes can be captured by amine groups on afunctionalized substrate. In other examples, an analyte (e.g., apolypeptide analyte) can be captured via an analyte binding moietydirectly attached to a substrate. In some embodiments, the substrate maybe populated with analyte minding moieties directly attached to thesubstrate as well as spatially-barcoded capture probes directly attachedto the substrate. In other embodiments, an analyte (e.g., a polypeptideanalyte) can be captured via an analyte binding moiety indirectlyattached to a substrate. In an example, the substrate may be populatedwith capture probes that are bound to an analyte capture agent, whereinthe analyte capture domain of the analyte capture agent binds to thecapture domain of the capture probe and the analyte binding moiety bindsthe polypeptide analyte.

In some embodiments, an analyte (e.g., a polypeptide analyte) can bedirectly captured or immobilized on a substrate. Direct immobilizationmay be achieved by covalently coupling the polypeptide analyte to thesubstrate via amide bonds between the carboxylic acid of the C-terminalamino acid residue and a functionalized substrate surface. For example,a substrate (e.g., a glass coverslip or slide) can be functionalizedthrough amino-silanization with aminopropyltriethoxysilane. Thesubstrate surfaces are further passivated by overnight incubation withpolyethylene glycol (PEG)-NHS solution, and functionalized slides can bestored in a vacuum desiccator until use. The t-butyloxycarbonylprotecting groups can be removed by incubating the substrate with 90%TFA (v/v in water) for 5 hours before use, thus exposing free aminegroups for peptide immobilization. The resulting functionalizedsubstrate is stable to multiple cycles of Edman degradation and washingsteps.

In some embodiments, methods for capturing polypeptides in a biologicalsample include providing a substrate where an analyte binding moiety isdirectly immobilized on the substrate. In some embodiments, directimmobilization is achieved through chemical modification of thesubstrate and/or chemical modification of the analyte binding moiety.For example, a substrate can be prepared with free amines on thesurface. When exposed to an analyte binding moiety with a freecarboxylic acid on the C-terminal residue, the free amines can formamide bonds with the carboxylic acid thereby covalently coupling theanalyte binding moiety to the substrate. Substrates and/or analytebinding moieties can be modified in any manner that facilitates covalentbonding of the analyte binding moiety to the substrate. Non-limitingexamples of chemical modification that can be used to covalently bindthe analyte binding moiety to the substrate include are describedherein.

In some embodiments, methods for capturing analyte polypeptides includeproviding a substrate (e.g., an array) where the analyte binding moietyis indirectly attached to the substrate. For example, an analyte bindingmoiety can be indirectly attached to a substrate via an oligonucleotide(e.g., a capture agent barcode domain or capture agent barcode domainhybridized to a capture probe) or other domain capable of binding toboth the substrate and the analyte binding domain. The capture agentbarcode domain is described elsewhere herein. The capture agent barcodedomain can be modified to include a cleavage domain, which can attach toa substrate using any of the chemistries described herein. In someembodiments, the capture agent barcode domain can include an analytecapture sequence as described herein, wherein the analyte capturesequence can hybridize to the capture domain of a capture probe. In someembodiments, a substrate (e.g., an array) containing capture probes canbe modified to capture polypeptide analytes by hybridizing the analytecapture sequence of the analyte capture agent to the capture domain of acapture probe.

In some embodiments, methods for capturing analyte polypeptides includeproviding a substrate (e.g., an array) and providing an analyte captureagent to the biological sample. For example, after drying and fixingsectioned tissue samples, the tissue samples can be positioned on asubstrate (e.g., a spatial array), rehydrated, blocked, andpermeabilized (e.g., 3XSSC, 2% BSA, 0.1% Triton X, 1 U/µl RNAseinhibitor for 10 min at 4° C.) before being stained with fluorescentprimary antibodies (1:100) and a pool of analyte capture agents (in3XSSC, 2% BSA, 0.1% Triton X, 1 U/µl RNAse inhibitor for 30 min at 4°C.). The biological sample can be washed, coverslipped (in glycerol + 1U/µl RNAse inhibitor), imaged for detected analytes (e.g., using aconfocal microscope or other apparatus capable of fluorescentdetection), and washed again. The analyte-bound analyte capture agentscan be released from the biological sample (e.g., the biological samplecan be treated with proteinase, e.g., proteinase K) and migrated to thespatial array. An analyte capture sequence of the analyte-bound analytecapture agent can be captured by a capture probe capture domain, and thecapture agent barcode domain can be extended to produce aspatially-tagged analyte capture agent. The spatially-tagged analytecapture agents can be processed according to spatial workflows describedherein.

In some embodiments, methods for capturing analyte polypeptides includeproviding blocking probes to analyte capture agents before introducingthe analyte capture agents to a biological sample. In some embodiments,the blocking probes can be alternatively or additionally provided in anyof the rehydrating or blocking buffers provided herein. In someembodiments, the analyte capture agent analyte capture sequence can beblocked prior to binding to the capture probe capture domain using ablocking probe sequence complementary to the analyte capture sequence.Blocking the capture agent barcode domain, particularly the free 3′ endof the capture agent barcode domain (e.g., analyte capture sequence),prior to contacting the analyte capture agents with the biologicalsample and/or substrate, can prevent binding of the analyte capturesequence of the capture agent barcode domains, e.g., prevents thebinding of a poly(A) tail to the capture probe capture domain. In someembodiments, blocking the analyte capture agent analyte capture domainreduces non-specific background staining. In some embodiments, theblocking probes are reversible, such that the blocking probes can beremoved from the analyte capture sequence during or after the time thatanalyte capture agents are in contact with the biological sample. Insome embodiments, the blocking probe can be removing with RNAsetreatment (e.g., RNAse H treatment).

In some embodiments, methods for capturing polypeptides in a biologicalsample include active transfer (e.g., electrophoresis). For example, thebiological sample is placed on a conductive substrate and contacted witha spatial array including one or more analyte binding moieties. Anelectric filed can be applied to the conductive substrate to promotemigration of the polypeptides towards the analyte binding moieties, asdescribed herein.

In some embodiments, methods for identifying the spatial location of apolypeptide in a biological sample include determining the sequence of acaptured polypeptide. In some embodiments, the sequence of the capturedpolypeptide is determined through detection of amino acid residueslabeled with a detectable label (e.g., radiolabel of a fluorophore).Non-limiting examples of detectable labels that can be used forlabelling the captured polypeptide include fluorophores and radiolabels.In some embodiments, the polypeptides are labeled at specific amino acidresidues only (e.g., not all amino acid residues are labeled). In someembodiments, the polypeptide is labeled prior to contacting thebiological sample with the substrate. In some embodiments, a capturedpolypeptide is labeled with fluorophores using standard coupling schemes(see Hernandez et al., New J. Chem. 41:462-469 (2017)). For example,polypeptides may be labeled by reaction with Atto647N-NHS,Atto647Niodoacetamide, TMR-NHS, or JF549-NHS, as appropriate, to labellysines (via NHS) or cysteines (via iodoacetamide). In addition, serineor threonine phosphorylation sites may be selectively labeled via betaelimination followed by conjugate addition via thiols to substitutethiol-linked fluorophores in place of phosphates (see Stevens et al.,Rapid Commun. Mass Specrtom., 15: 2157-2162 (2005)). The number offluorophores incorporated into a polypeptide is any number that may bespectrally resolved. In some instances, four or more fluorophores areutilized.

In some embodiments, a captured polypeptide is radiolabeled. In someembodiments, specific amino acids can be labeled with an isotope.Non-limiting examples of isotopes used to label amino acids include ³H,¹⁴C, ¹⁵N, ³²P, and ¹²⁵I. In some embodiments, the isotope isincorporated into the selected amino acid prior to incorporation into apolypeptide. In some embodiments, the radiolabeled amino acid can beincorporated into the polypeptide after polypeptide formation.

In some embodiments, the sequence of the captured polypeptide isdetermined using Edman degradation (and in some embodiments successiverounds of Edman degradation). In such cases, a polypeptide is captured,and the polypeptide sequence can be resolved by imaging the substratefollowing repeated rounds of Edman degradation. For example, thesubstrate is imaged following each Edman reaction in order to capturethe detectable labels that are produced due to the removal of aminoacids that are a byproduct of the reaction. The information obtained bythe Edman degradation can be complied to identify a polypeptide. In someembodiments, the biological sample is visualized or imaged using lightor fluorescence microscopy.

(VIII) Enrichment of Captured Analytes After Capture

In some aspects, spatial analysis of targeted analytes includes anenrichment step or steps post-capture to enrich the captured analytesfor the targeted analyte. For example, the capture domain can beselected or designed for the selective capture of more analytes than thepractitioner desires to analyze. In some embodiments, capture probesthat include random sequences (e.g., random hexamers or similarsequences) that form all or part of the capture domain can be used tocapture nucleic acids from a biological sample in an unbiased way. Forexample, capture probes having capture domains that include randomsequences can be used to generically capture DNA, RNA, or both from abiological sample. Alternatively, capture probes can include capturedomains can that capture mRNA generally. As is well known in the art,this can be on the basis of hybridization to the poly-A tail of mRNAs.In some embodiments, the capture domain includes a sequence thatinteracts with (e.g., hybridizes to) the poly-A tail of mRNAs.Non-limiting examples of such sequences include poly-T DNA sequences andpoly-U RNA sequences. In some embodiments, random sequences can be usedin conjunction with poly-T (or poly-T analogue etc.) sequences. Thus,where a capture domain includes a poly-T (or a “poly-T-like”)oligonucleotide, it can also include a random oligonucleotide sequence.

In some aspects, after capture of more analytes than the practitionerdesires to analyze, methods disclosed herein include enrichment ofparticular captured analytes. In some aspects, methods includeenrichment of analytes that include mutations (e.g., SNPs,) of interest,nucleic acid(s) of interest, and/or proteins(s) of interest.

In some embodiments, methods of spatial analysis provided herein includeselectively enriching one or more analytes of interest (e.g., targetanalytes) after analyte capture. For example, one or more analytes ofinterest can be enriched by addition of one or more oligonucleotides tothe pool of captured analytes. In some embodiments, one or more analytesof interest can be enriched by addition of one or more oligonucleotidesto the pool of captured analytes on the array. In some embodiments, oneor more analytes of interest can be enriched by addition of one or moreoligonucleotides to the pool of captured analytes where the pool ofcaptured analytes have been released (e.g., removed) from the array. Insome embodiments, when captured analytes have been released from thearray the one or more nucleotides can be complementary to a portion of aTSO and R1 sequence, or portion thereof. In some embodiments, theadditional oligonucleotide(s) include a sequence used for priming areaction by a polymerase. For example, one or more primer sequences withsequence complementarity to one or more analytes of interest can be usedto amplify the one or more analyte(s) of interest, thereby selectivelyenriching these analytes. In some embodiments, one or more primersequences can be complementary to other domains on the capture probe(e.g., R1 sequence, or portion thereof, as above), and not complementaryto the analyte. In some embodiments, enrichment by amplification (e.g.,PCR) occurs by using a first primer complementary to an analyte oranalytes of interest (or another domain in the capture probe and theTSO), or complement thereof, and a second primer complementary to aregion of the capture probe, or complement thereof. In some embodiments,the region of the capture probe, or complement thereof, is distal to aspatial barcode from the capture domain, such that enrichment byamplification amplifies both the captured analyte or analytes and its ortheir associated spatial barcodes, thus permitting spatial analysis ofthe enriched analyte or analytes.

In some embodiments, two or more capture probes capture two or moredistinct analytes, which analytes are enriched (e.g., simultaneously orsequentially) from the pool of captured analytes. In some embodiments,enrichment by PCR amplification includes multiple rounds ofamplification. For example, enrichment by PCR amplification can includenested PCR reactions using different primers that are specific for theanalyte or analytes of interest. In some embodiments, enrichment byamplification can be performed using an amplification method that is notPCR. A non-limiting example of a non-PCR amplification method is rollingcircle amplification. Other non-PCR amplification methods are known inthe art.

In some embodiments, an oligonucleotide with sequence complementarity toa captured analyte or analytes of interest, or complement thereof, canbe used to enrich the captured analyte or analytes of interest from thepool of captured analytes. In some embodiments, an oligonucleotide withsequence complementarity to a captured analyte or analytes of interest(or another domain the capture probe), or complement thereof, caninclude one or more functional moieties that are useful in theenrichment process. For example, biotinylated oligonucleotides withsequence complementary to one or more analytes interest, or complementsthereof, can bind to the analyte(s) of interest and can be selectedusing biotinylation-strepavidin affinity using any of a variety ofmethods known in the art (e.g., streptavidin beads). In someembodiments, oligonucleotides with sequence complementary to one or moreanalytes interest, or complements thereof, include a magnetic moiety(e.g., a magnetic bead) that can be used in the enrichment process.

Additionally or alternatively, one or more species of analyte (e.g.,mitochondrial DNA or RNA) can be down-selected (e.g., removed) using anyof a variety of methods. In some embodiments, such down-selection ofanalytes that are not of interest can result in improved capture ofother types of analytes that are of interest. For example, probes can beadministered to a sample that selectively hybridize to ribosomal RNA(rRNA), thereby reducing the pool and concentration of rRNA in thesample. In some embodiments, such down-selection can result in improvedcapture of other types of RNA due to the reduction in non-specific RNApresent in the sample. Additionally or alternatively, duplex-specificnuclease (DSN) treatment can remove rRNA (see, e.g., Archer, et al,Selective and flexible depletion of problematic sequences from RNA-seqlibraries at the cDNA stage, BMC Genomics, 15 401, (2014), the entirecontents of which is incorporated herein by reference). In someembodiments, hydroxyapatite chromatography can be used to removeabundant species (e.g., rRNA).

(IX) RNA-Templated Ligation

In some embodiments of methods provided here, RNA-templated ligation isused to interrogate spatial gene expression in a biological sample(e.g., an FFPE tissue section). RNA-templated ligation enables sensitivemeasurement of specific nucleic acid analytes of interest that otherwisemight be analyzed less sensitively with a whole transcriptomic approach.It provides advantages of compatibility with common histochemical stainsand suitability for analysis of decade-old materials (e.g., FFPEsamples) and exceedingly small microdissected tissue fragments.

In some aspects, the steps of RNA-templated ligation include: (1)hybridization of pairs of probes (e.g., DNA probes) to RNA (e.g.,formalin fixed RNA) within a tissue section; (2) ligation of adjacentlyannealed probe pairs in situ; (3) RNase H treatment that (i) releasesRNA-templated ligation products from the tissue (e.g., into solution)for downstream analysis and (ii) destroys unwanted DNA-templatedligation products; and optionally, (4) amplification of RNA -templatedligation products (e.g., by multiplex PCR).

In some aspects, disclosed herein are methods of direct detection of RNAtarget-DNA probe duplexes without first converting RNA to cDNA byreverse transcription. In some aspects, RNA-templated ligation caninclude a DNA ligase. In some aspects, RNA-templated ligation caninclude RNA ligase. In some aspects, RNA-templated ligation can includeT4 RNA ligase.

In some aspects, RNA-templated ligation is used for detection of RNA,determination of RNA sequence identity, and/or expression monitoring andtranscript analysis. In some aspects, RNA-templated ligation allows fordetection of a particular change in a nucleic acid (e.g., a mutation orsingle nucleotide polymorphism (SNP)), detection or expression of aparticular nucleic acid, or detection or expression of a particular setof nucleic acids (e.g., in a similar cellular pathway or expressed in aparticular pathology). In some embodiments, the methods that includeRNA-templated ligation are used to analyze nucleic acids, e.g., bygenotyping, quantitation of DNA copy number or RNA transcripts,localization of particular transcripts within samples, and the like. Insome aspects, systems and methods provided herein that includeRNA-templated ligation identify single nucleotide polymorphisms (SNPs).In some aspects, such systems and methods identify mutations.

In some aspects, disclosed herein are methods of detecting RNAexpression that include bringing into contact a first probe, a secondprobe, and ligase (e.g., T4 RNA ligase). In some embodiments, the firstprobe and the second probe are designed to hybridize to a targetsequence such that the 5′ end of the first probe and the 3′ end of thesecond probe are adjacent and can be ligated, wherein at least the5′-terminal nucleotide of the first probe and at least the 3′-terminalnucleotide of the second probe are deoxyribonucleotides (DNA), andwherein the target sequence includes (e.g., is composed of)ribonucleotides (RNA). After hybridization, a ligase (e.g., T4 RNAligase) ligates the first probe and the second probe if the targetsequence is present in the target sample, but does not ligate the firstprobe and the second probe if the target sequence is not present in thetarget sample. The presence or absence of the target sequence in thebiological sample can be determined by determining whether or not thefirst and second probes were ligated in the presence of ligase. Any of avariety of methods can be used to determine whether or not the first andsecond probes were ligated in the presence of ligase, including but notlimited to, sequencing the ligated product, hybridizing the ligatedproduct with a detection probe that hybridizes only when the first andsecond probes were ligated in the presence of ligase, restriction enzymeanalysis, and other methods known in the art.

In some aspects, two or more RNA analytes are analyzed using methodsthat include RNA-templated ligation. In some aspects, when two or moreanalytes are analyzed, a first and second probe that is specific for(e.g., specifically hybridizes to) each RNA analyte are used.

In some aspects, three or more probes are used in RNA-templated ligationmethods provided herein. In some embodiments, the three or more probesare designed to hybridize to a target sequence such that the three ormore probes hybridize adjacent to each other such that the 5′ and 3′ends of adjacent probes can be ligated. In some embodiments, thepresence or absence of the target sequence in the biological sample canbe determined by determining whether or not the three or more probeswere ligated in the presence of ligase.

In some aspects, the first probe is a DNA probe. In some aspects, thefirst probe is a chimeric DNA/RNA probe. In some aspects, the secondprobe is a DNA probe. In some aspects, the second probe is a chimericDNA/RNA probe.

In some aspects, methods of RNA-templated ligation utilize the T4 RNALigase 2 to efficiently join adjacent chimeric RNA-DNA probe pairshybridized in situ on fixed RNA target sequences. Subsequent treatmentwith RNase H releases RNA-templated ligation products (e.g., intosolution) for downstream analysis.

(X) Region of Interest

A biological sample can have regions that show morphological feature(s)that may indicate the presence of disease or the development of adisease phenotype. For example, morphological features at a specificsite within a tumor biopsy sample can indicate the aggressiveness,therapeutic resistance, metastatic potential, migration, stage,diagnosis, and/or prognosis of cancer in a subject. A change in themorphological features at a specific site within a tumor biopsy sampleoften correlate with a change in the level or expression of an analytein a cell within the specific site, which can, in turn, be used toprovide information regarding the aggressiveness, therapeuticresistance, metastatic potential, migration, stage, diagnosis, and/orprognosis of cancer in a subject. A region or area within a biologicalsample that is selected for specific analysis (e.g., a region in abiological sample that has morphological features of interest) is oftendescribed as “a region of interest.”

A region of interest in a biological sample can be used to analyze aspecific area of interest within a biological sample, and thereby, focusexperimentation and data gathering to a specific region of a biologicalsample (rather than an entire biological sample). This results inincreased time efficiency of the analysis of a biological sample.

A region of interest can be identified in a biological sample using avariety of different techniques, e.g., expansion microscopy, brightfield microscopy, dark field microscopy, phase contrast microscopy,electron microscopy, fluorescence microscopy, reflection microscopy,interference microscopy, confocal microscopy, and visual identification(e.g., by eye), and combinations thereof. For example, the staining andimaging of a biological sample can be performed to identify a region ofinterest. In some examples, the region of interest can correspond to aspecific structure of cytoarchitecture. In some embodiments, abiological sample can be stained prior to visualization to providecontrast between the different regions of the biological sample. Thetype of stain can be chosen depending on the type of biological sampleand the region of the cells to be stained. In some embodiments, morethan one stain can be used to visualize different aspects of thebiological sample, e.g., different regions of the sample, specific cellstructures (e.g., organelles), or different cell types. In otherembodiments, the biological sample can be visualized or imaged withoutstaining the biological sample.

In some embodiments, staining and imaging a biological sample prior tocontacting the biological sample with a spatial array is performed toselect samples for spatial analysis. In some embodiments, the stainingincludes applying a fiducial marker as described herein, includingfluorescent, radioactive, chemiluminescent, or colorimetric detectablemarkers. In some embodiments, the staining and imaging of biologicalsamples allows the user to identify the specific sample (or region ofinterest) the user wishes to assess.

In some examples, an array (e.g., any of the exemplary arrays describedherein) can be contacted with only a portion of a biological sample(e.g., a cell, a tissue section, or a region of interest). In someexamples, a biological sample is contacted with only a portion of anarray (e.g., any of the exemplary arrays described herein). In someembodiments, capture probes on an array corresponding to regions ofinterest of a biological sample (e.g., proximal to the region ofinterest) can be selectively cleaved and analyzed. For example, captureprobes on an array may be deactivated or eliminated outside of areascorresponding to regions of interest of a biological sample. In someembodiments, capture probes including a photocleavable bond and on thearray in areas corresponding to regions of interest of a biologicalsample can be selectively cleaved by using light. A mirror, mirrorarray, a lens, a moving stage, and/or a photomask can be used to directthe light to regions of the array that correspond areas outside one ormore regions of interest in the biological sample. Some embodimentsinclude deactivating or eliminating capture probes, e.g., capture probescomprising a photocleavable bond as described herein, using light. Insome embodiments, a laser, e.g., a scanning laser, can be used todeactivate or eliminate capture probes. In some embodiments, theeliminated member of the plurality of capture probes can be washed away.In some embodiments, regions of interest can be labeled with differentheavy metals, and a laser can sequentially ablate these regions ofinterest before mass spectrometry identification. A laser can, forexample, deactivate or eliminate capture probes through UV lightdestruction of DNA, heat, inducing a chemical reaction that prevents thecapture probes from moving to the next step, inducing photocleavage of aphotocleavable bond, or a combination thereof. In some examples, aportion of the array can be deactivated such that it does not interactwith the analytes in the biological sample (e.g., optical deactivation,chemical deactivation, heat deactivation, or blocking of the captureprobes in the array (e.g., using blocking probes)). In some examples, aregion of interest can be removed from a biological sample and then theregion of interest can be contacted to the array (e.g., any of thearrays described herein). A region of interest can be removed from abiological sample using microsurgery, laser capture microdissection,chunking, a microtome, dicing, trypsinization, labelling, and/orfluorescence-assisted cell sorting, and the like.

In some examples, a region of interest can be permeabilized or lysedwhile areas outside the region of interest are not permeabilized orlysed (e.g., Kashyap et al. Sci Rep. 2016; 6: 29579, herein incorporatedby reference in its entirety). For example, in some embodiments, aregion of interest can be contacted with a hydrogel comprising apermeabilization or lysing reagent. In some embodiments, the area(s)outside the region of interest are not contacted with the hydrogelcomprising the permeabilization or lysing reagent.

(F) Partitioning

As discussed above, in some embodiments, the sample can optionally beseparated into single cells, cell groups, or other fragments/pieces thatare smaller than the original, unfragmented sample. Each of thesesmaller portions of the sample can be analyzed to obtainspatially-resolved analyte information for the sample.

For samples that have been separated into smaller fragments - andparticularly, for samples that have been disaggregated, dissociated, orotherwise separated into individual cells - one method for analyzing thefragments involves separating the fragments into individual partitions(e.g., fluid droplets), and then analyzing the contents of thepartitions. In general, each partition maintains separation of its owncontents from the contents of other partitions. The partition can be adroplet in an emulsion, for example.

The partitions can be flowable within fluid streams. The partitions caninclude, for example, micro-vesicles that have an outer barriersurrounding an inner fluid center or core. In some cases, the partitionscan include a porous matrix that is capable of entraining and/orretaining materials within its matrix. The partitions can be droplets ofa first phase within a second phase, wherein the first and second phasesare immiscible. For example, the partitions can be droplets of aqueousfluid within a non-aqueous continuous phase (e.g., oil phase). Inanother example, the partitions can be droplets of a non-aqueous fluidwithin an aqueous phase. In some examples, the partitions can beprovided in a water-in-oil emulsion or oil-in-water emulsion. A varietyof different vessels are described in, for example, U.S. Pat.Application Publication No. 2014/0155295, the entire contents of whichare incorporated herein by reference. Emulsion systems for creatingstable droplets in non-aqueous or oil continuous phases are described,for example, in U.S. Pat. Application Publication No. 2010/0105112, theentire contents of which are incorporated herein by reference.

For droplets in an emulsion, allocating individual particles to discretepartitions can be accomplished, for example, by introducing a flowingstream of particles in an aqueous fluid into a flowing stream of anon-aqueous fluid, such that droplets are generated at the junction ofthe two streams. Fluid properties (e.g., fluid flow rates, fluidviscosities, etc.), particle properties (e.g., volume fraction, particlevolume, particle concentration, etc.), microfluidic architectures (e.g.,channel geometry, etc.), and other parameters can be adjusted to controlthe occupancy of the resulting partitions (e.g., number of analytes perpartition, number of beads per partition, etc.). For example, partitionoccupancy can be controlled by providing the aqueous stream at a certainconcentration and/or flow rate of analytes.

To generate single analyte partitions, the relative flow rates of theimmiscible fluids can be selected such that, on average, the partitionscan contain less than one analyte per partition to ensure that thosepartitions that are occupied are primarily singly occupied. In somecases, partitions among a plurality of partitions can contain at mostone analyte. In some embodiments, the various parameters (e.g., fluidproperties, particle properties, microfluidic architectures, etc.) canbe selected or adjusted such that a majority of partitions are occupied,for example, allowing for only a small percentage of unoccupiedpartitions. The flows and channel architectures can be controlled as toensure a given number of singly occupied partitions, less than a certainlevel of unoccupied partitions and/or less than a certain level ofmultiply occupied partitions.

The channel segments described herein can be coupled to any of a varietyof different fluid sources or receiving components, includingreservoirs, tubing, manifolds, or fluidic components of other systems.As will be appreciated, the microfluidic channel structure can have avariety of geometries. For example, a microfluidic channel structure canhave one or more than one channel junction. As another example, amicrofluidic channel structure can have 2, 3, 4, or 5 channel segmentseach carrying particles that meet at a channel junction. Fluid can bedirected to flow along one or more channels or reservoirs via one ormore fluid flow units. A fluid flow unit can include compressors (e.g.,providing positive pressure), pumps (e.g., providing negative pressure),actuators, and the like to control flow of the fluid. Fluid can also orotherwise be controlled via applied pressure differentials, centrifugalforce, electrokinetic pumping, vacuum, capillary, and/or gravity flow.

In addition to cells and/or analytes, a partition can include additionalcomponents, and in particular, one or more beads. A partition caninclude a single gel bead, a single cell bead, or both a single cellbead and single gel bead. A variety of different beads can beincorporated into partitions. In some embodiments, for example,non-barcoded beads can be incorporated into the partitions. For example,where the biological particle (e.g., a cell) that is incorporated intothe partitions carries one or more barcodes (e.g., spatial barcode(s),UMI(s), and combinations thereof), the bead can be a non-barcoded bead.

In some embodiments, a barcode carrying bead can be incorporated intopartitions. In general, an individual bead can be coupled to any numberof individual nucleic acid molecules, for example, from one to tens tohundreds of thousands or even millions of individual nucleic acidmolecules. The respective barcodes for the individual nucleic acidmolecules can include both common sequence segments or relatively commonsequence segments and variable or unique sequence segments betweendifferent individual nucleic acid molecules coupled to the same bead.For example, a nucleic acid molecule (e.g., an oligonucleotide), can becoupled to a bead by a releasable linkage (e.g., a disulfide linker),wherein the nucleic acid molecule can be or include a barcode. Forexample, barcodes can be injected into droplets previous to, subsequentto, or concurrently with droplet generation. The delivery of thebarcodes to a particular partition allows for the later attribution ofthe characteristics of the individual biological particle to theparticular partition. Barcodes can be delivered, for example on anucleic acid molecule (e.g., an oligonucleotide), to a partition via anysuitable mechanism. Barcoded nucleic acid molecules can be delivered toa partition via a microcapsule. A microcapsule, in some instances, caninclude a bead. The same bead can be coupled (e.g., via releasablelinkage) to one or more other nucleic acid molecules.

The nucleic acid molecule can include a functional domain that can beused in subsequent processing. For example, the functional domain caninclude one or more of a sequencer specific flow cell attachmentsequence (e.g., a P5 sequence for Illumina® sequencing systems) and asequencing primer sequence (e.g., a R1 primer for Illumina® sequencingsystems). The nucleic acid molecule can include a barcode sequence foruse in barcoding the sample (e.g., DNA, RNA, protein, etc.). In somecases, the barcode sequence can be bead-specific such that the barcodesequence is common to all nucleic acid molecules coupled to the samebead. Alternatively or in addition, the barcode sequence can bepartition-specific such that the barcode sequence is common to allnucleic acid molecules coupled to one or more beads that are partitionedinto the same partition. The nucleic acid molecule can include aspecific priming sequence, such as an mRNA specific priming sequence(e.g., poly(T) sequence), a targeted priming sequence, and/or a randompriming sequence. The nucleic acid molecule can include an anchoringsequence to ensure that the specific priming sequence hybridizes at thesequence end (e.g., of the mRNA). For example, the anchoring sequencecan include a random short sequence of nucleotides, such as a 1-mer,2-mer, 3-mer or longer sequence, which can ensure that a poly(T) segmentis more likely to hybridize at the sequence end of the poly(A) tail ofthe mRNA.

The nucleic acid molecule can include a unique molecular identifyingsequence (e.g., unique molecular identifier (UMI)). In some embodiments,the unique molecular identifying sequence can include from about 5 toabout 8 nucleotides. Alternatively, the unique molecular identifyingsequence can include less than about 5 or more than about 8 nucleotides.The unique molecular identifying sequence can be a unique sequence thatvaries across individual nucleic acid molecules coupled to a singlebead. In some embodiments, the unique molecular identifying sequence canbe a random sequence (e.g., such as a random N-mer sequence). Forexample, the UMI can provide a unique identifier of the starting mRNAmolecule that was captured, in order to allow quantitation of the numberof original expressed RNA.

A partition can also include one or more reagents. Unique identifiers,such as barcodes, can be injected into the droplets previous to,subsequent to, or concurrently with droplet generation, such as via amicrocapsule (e.g., bead). Microfluidic channel networks (e.g., on achip) can be utilized to generate partitions. Alternative mechanisms canalso be employed in the partitioning of individual biological particles,including porous membranes through which aqueous mixtures of cells areextruded into non-aqueous fluids.

In some embodiments, barcoded nucleic acid molecules can be initiallyassociated with a microcapsule and then released from the microcapsule.Release of the barcoded nucleic acid molecules can be passive (e.g., bydiffusion out of the microcapsule). In addition or alternatively,release from the microcapsule can be upon application of a stimuluswhich allows the barcoded nucleic acid nucleic acid molecules todissociate or to be released from the microcapsule. Such stimulus candisrupt the microcapsule, an interaction that couples the barcodednucleic acid molecules to or within the microcapsule, or both. Suchstimulus can include, for example, a thermal stimulus, photo-stimulus,chemical stimulus (e.g., change in pH or use of a reducing agent(s)), amechanical stimulus, a radiation stimulus; a biological stimulus (e.g.,enzyme), or any combination thereof.

In some embodiments, one more barcodes (e.g., spatial barcodes, UMIs, ora combination thereof) can be introduced into a partition as part of theanalyte. As described previously, barcodes can be bound to the analytedirectly, or can form part of a capture probe or analyte capture agentthat is hybridized to, conjugated to, or otherwise associated with ananalyte, such that when the analyte is introduced into the partition,the barcode(s) are introduced as well. As described above, FIG. 16 showsan example of a microfluidical channel structure for partitioningindividual analytes (e.g., cells) into discrete partitions.

FIG. 16 shows an example of a microfluidic channel structure forpartitioning individual analytes (e.g., cells) into discrete partitions.The channel structure can include channel segments 1601, 1602, 1603, and1604 communicating at a channel junction 1605. In operation, a firstaqueous fluid 1606 that includes suspended biological particles (orcells) 1607 may be transported along channel segment 1601 into junction1605, while a second fluid 1608 that is immiscible with the aqueousfluid 1606 is delivered to the junction 1605 from each of channelsegments 1602 and 1603 to create discrete droplets 1609, 1610 of thefirst aqueous fluid 1606 flowing into channel segment 1604, and flowingaway from junction 1605. The channel segment 1604 may be fluidicallycoupled to an outlet reservoir where the discrete droplets can be storedand/or harvested. A discrete droplet generated may include an individualbiological particle 1607 (such as droplets 1609). A discrete dropletgenerated may include more than one individual biological particle 1607.A discrete droplet may contain no biological particle 1607 (such asdroplet 1610). Each discrete partition may maintain separation of itsown contents (e.g., individual biological particle 1607) from thecontents of other partitions.

FIG. 17A shows another example of a microfluidic channel structure 1700for delivering beads to droplets. The channel structure includes channelsegments 1701, 1702, 1703, 1704 and 1705 communicating at a channeljunction 1706. During operation, the channel segment 1701 can transportan aqueous fluid 1707 that includes a plurality of beads 1708 along thechannel segment 1701 into junction 1706. The plurality of beads 1708 canbe sourced from a suspension of beads. For example, the channel segment1701 can be connected to a reservoir that includes an aqueous suspensionof beads 1708. The channel segment 1702 can transport the aqueous fluid1707 that includes a plurality of particles 1709 (e.g., cells) along thechannel segment 1702 into junction 1706. In some embodiments, theaqueous fluid 1707 in either the first channel segment 1701 or thesecond channel segment 1702, or in both segments, can include one ormore reagents, as further described below.

A second fluid 1710 that is immiscible with the aqueous fluid 1707(e.g., oil) can be delivered to the junction 1706 from each of channelsegments 1703 and 1704. Upon meeting of the aqueous fluid 1707 from eachof channel segments 1701 and 1702 and the second fluid 1710 from each ofchannel segments 1703 and 1704 at the channel junction 1706, the aqueousfluid 1707 can be partitioned as discrete droplets 1711 in the secondfluid 1710 and flow away from the junction 1706 along channel segment1705. The channel segment 1705 can deliver the discrete droplets to anoutlet reservoir fluidly coupled to the channel segment 1705, where theycan be harvested.

As an alternative, the channel segments 1701 and 1702 can meet atanother junction upstream of the junction 1706. At such junction, beadsand biological particles can form a mixture that is directed alonganother channel to the junction 1706 to yield droplets 1711. The mixturecan provide the beads and biological particles in an alternatingfashion, such that, for example, a droplet includes a single bead and asingle biological particle.

The second fluid 1710 can include an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets1711.

The partitions described herein can include small volumes, for example,less than about 10 microliters (µL), 5 µL, 1 µL, 900 picoliters (pL),800 pL, 700 pL, 600 pL, 500 pL, 400pL, 300 pL, 200 pL, 100pL, 50 pL, 20pL, 10 pL, 1 pL, 500 nanoliters (nL), 100 nL, 50 nL, or less.

In the foregoing discussion, droplets with beads were formed at thejunction of different fluid streams. In some embodiments, droplets canbe formed by gravity-based partitioning methods.

FIG. 17B shows a cross-section view of another example of a microfluidicchannel structure 1750 with a geometric feature for controlledpartitioning. A channel structure 1750 can include a channel segment1752 communicating at a channel junction 1758 (or intersection) with areservoir 1754. In some instances, the channel structure 1750 and one ormore of its components can correspond to the channel structure 1700 andone or more of its components.

An aqueous fluid 1760 comprising a plurality of particles 1756 may betransported along the channel segment 1752 into the junction 1758 tomeet a second fluid 1762 (e.g., oil, etc.) that is immiscible with theaqueous fluid 1760 in the reservoir 1754 to create droplets 1764 of theaqueous fluid 1760 flowing into the reservoir 1754. At the junction 1758where the aqueous fluid 1760 and the second fluid 1762 meet, dropletscan form based on factors such as the hydrodynamic forces at thejunction 1758, relative flow rates of the two fluids 1760, 1762, fluidproperties, and certain geometric parameters (e.g., Δh, etc.) of thechannel structure 1750. A plurality of droplets can be collected in thereservoir 1754 by continuously injecting the aqueous fluid 1760 from thechannel segment 1752 at the junction 1758.

A discrete droplet generated may comprise one or more particles of theplurality of particles 1756. As described elsewhere herein, a particlemay be any particle, such as a bead, cell bead, gel bead, biologicalparticle, macromolecular constituents of biological particle, or otherparticles. Alternatively, a discrete droplet generated may not includeany particles.

In some instances, the aqueous fluid 1760 can have a substantiallyuniform concentration or frequency of particles 1756. As describedelsewhere herein, the particles 1756 (e.g., beads) can be introducedinto the channel segment 1752 from a separate channel (not shown in FIG.17 ). The frequency of particles 1756 in the channel segment 1752 may becontrolled by controlling the frequency in which the particles 1756 areintroduced into the channel segment 1752 and/or the relative flow ratesof the fluids in the channel segment 1752 and the separate channel. Insome instances, the particles 1756 can be introduced into the channelsegment 1752 from a plurality of different channels, and the frequencycontrolled accordingly. In some instances, different particles may beintroduced via separate channels. For example, a first separate channelcan introduce beads and a second separate channel can introducebiological particles into the channel segment 1752. The first separatechannel introducing the beads may be upstream or downstream of thesecond separate channel introducing the biological particles.

In some instances, the second fluid 1762 may not be subjected to and/ordirected to any flow in or out of the reservoir 1754. For example, thesecond fluid 1762 may be substantially stationary in the reservoir 1754.In some instances, the second fluid 1762 may be subjected to flow withinthe reservoir 1754, but not in or out of the reservoir 1754, such as viaapplication of pressure to the reservoir 1754 and/or as affected by theincoming flow of the aqueous fluid 1760 at the junction 1758.Alternatively, the second fluid 1762 may be subjected and/or directed toflow in or out of the reservoir 1754. For example, the reservoir 1754can be a channel directing the second fluid 1762 from upstream todownstream, transporting the generated droplets.

The channel structure 1750 at or near the junction 1758 may have certaingeometric features that at least partly determine the volumes and/orshapes of the droplets formed by the channel structure 1750. The channelsegment 1752 can have a first cross-section height, h1, and thereservoir 1754 can have a second cross-section height, h2. The firstcross-section height, h1, and the second cross-section height, h2, maybe different, such that at the junction 1758, there is a heightdifference of Δh. The second cross-section height, h2, may be greaterthan the first cross-section height, h1. In some instances, thereservoir may thereafter gradually increase in cross-section height, forexample, the more distant it is from the junction 1758. In someinstances, the cross-section height of the reservoir may increase inaccordance with expansion angle, β, at or near the junction 1758. Theheight difference, Δh, and/or expansion angle, β, can allow the tongue(portion of the aqueous fluid 1760 leaving channel segment 1752 atjunction 1758 and entering the reservoir 1754 before droplet formation)to increase in depth and facilitate decrease in curvature of theintermediately formed droplet. For example, droplet volume may decreasewith increasing height difference and/or increasing expansion angle.

The height difference, Δh, can be at least about 1 µm. Alternatively,the height difference can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60,70, 80, 90, 100, 200, 300, 400, 500 µm or more. Alternatively, theheight difference can be at most about 500, 400, 300, 200, 100, 90, 80,70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, 1 µm or less. In some instances, theexpansion angle, β, may be between a range of from about 0.5° to about4°, from about 0.1° to about 10°, or from about 0° to about 90°. Forexample, the expansion angle can be at least about 0.01°, 0.1°, 0.2°,0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 6°, 7°,8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°,75°, 80°, 85°, or higher. In some instances, the expansion angle can beat most about 89°, 88°, 87°, 86°, 85°, 84°, 83°, 82°, 81°, 80°, 75°,70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15°, 10°, 9°, 8°,7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less.

In some instances, the flow rate of the aqueous fluid 1760 entering thejunction 1758 can be between about 0.04 microliters (µL)/minute (min)and about 40 µL/min. In some instances, the flow rate of the aqueousfluid 1760 entering the junction 1758 can be between about 0.01microliters (µL)/minute (min) and about 100 µL/min. Alternatively, theflow rate of the aqueous fluid 1760 entering the junction 1758 can beless than about 0.01 µL/min. alternatively, the flow rate of the aqueousfluid 1760 entering the junction 1758 can be greater than about 40µL/min, such as 45 µL/min, 50 µL/min, 55 µL/min, 60 µL/min, 65 µL/min,70 µL/min, 75 µL/min, 80 µL/min, 85 µL/min, 90 µL/min, 95 µL/min, 100µL/min, 110 µL/min, 120 µL/min, 130 µL/min, 140 µL/min, 150 µL/min, orgreater. At lower flow rates, such as flow rates of about less than orequal to 10 microliters/minute, the droplet radius may not be dependenton the flow rate of the aqueous fluid 1760 entering the junction 1758.The second fluid 1762 may be stationary, or substantially stationary, inthe reservoir 1754. Alternatively, the second fluid 1762 may be flowing,such as at the above flow rates described for the aqueous fluid 1760.

While FIG. 17B illustrates the height difference, Δh, being abrupt atthe junction 1758 (e.g., a step increase), the height difference mayincrease gradually (e.g., from about 0 µm to a maximum heightdifference). Alternatively, the height difference may decrease gradually(e.g., taper) from a maximum height difference. A gradual increase ordecrease in height difference, as used herein, may refer to a continuousincremental increase or decrease in height difference, wherein an anglebetween any one differential segment of a height profile and animmediately adjacent differential segment of the height profile isgreater than 90°. For example, at the junction 1758, a bottom wall ofthe channel and a bottom wall of the reservoir can meet at an anglegreater than 90°. Alternatively or in addition, a top wall (e.g.,ceiling) of the channel and a top wall (e.g., ceiling) of the reservoircan meet an angle greater than 90°. A gradual increase or decrease maybe linear or non-linear (e.g., exponential, sinusoidal, etc.).Alternatively or in addition, the height difference may variablyincrease and/or decrease linearly or non-linearly. While FIG. 17Billustrates the expanding reservoir cross-section height as linear(e.g., constant expansion angle, β), the cross-section height may expandnon-linearly. For example, the reservoir may be defined at leastpartially by a dome-like (e.g., hemispherical) shape having variableexpansion angles. The cross-section height may expand in any shape.

FIG. 17C depicts a workflow wherein cells are partitioned into dropletsalong with barcode-bearing beads 1770. See FIG. 17A. The droplet formsan isolated reaction chamber wherein the cells can be lysed 1771 andtarget analytes within the cells can then be captured 1772 and amplified1773, 1774 according to previously described methods. After sequencelibrary preparation clean-up 1775, the material is sequenced and/orquantified 1776 according to methods described herein.

It should be noted that while the example workflow in FIG. 17C includessteps specifically for the analysis of mRNA, analogous workflows can beimplemented for a wide variety of other analytes, including any of theanalytes described previously.

By way of example, in the context of analyzing sample RNA as shown inFIG. 17C, the poly(T) segment of one of the released nucleic acidmolecules (e.g., from the bead) can hybridize to the poly(A) tail of amRNA molecule. Reverse transcription can result in a cDNA transcript ofthe mRNA, which transcript includes each of the sequence segments of thenucleic acid molecule. If the nucleic acid molecule includes ananchoring sequence, it will more likely hybridize to and prime reversetranscription at the sequence end of the poly(A) tail of the mRNA.

Within any given partition, all of the cDNA transcripts of theindividual mRNA molecules can include a common barcode sequence segment.However, the transcripts made from the different mRNA molecules within agiven partition can vary at the unique molecular identifying sequencesegment (e.g., UMI segment). Beneficially, even following any subsequentamplification of the contents of a given partition, the number ofdifferent UMIs can be indicative of the quantity of mRNA originatingfrom a given partition. As noted above, the transcripts can beamplified, cleaned up and sequenced to identify the sequence of the cDNAtranscript of the mRNA, as well as to sequence the barcode segment andthe UMI segment. While a poly(T) primer sequence is described, othertargeted or random priming sequences can also be used in priming thereverse transcription reaction. Likewise, although described asreleasing the barcoded oligonucleotides into the partition, in somecases, the nucleic acid molecules bound to the bead can be used tohybridize and capture the mRNA on the solid phase of the bead, forexample, in order to facilitate the separation of the RNA from othercell contents.

In some embodiments, partitions include precursors that include afunctional group that is reactive or capable of being activated suchthat it becomes reactive can be polymerized with other precursors togenerate gel beads that include the activated or activatable functionalgroup. The functional group can then be used to attach additionalspecies (e.g., disulfide linkers, primers, other oligonucleotides, etc.)to the gel beads. For example, some precursors featuring a carboxylicacid (COOH) group can co-polymerize with other precursors to form a beadthat also includes a COOH functional group. In some cases, acrylic acid(a species comprising free COOH groups), acrylamide, andbis(acryloyl)cystamine can be copolymerized together to generate a beadwith free COOH groups. The COOH groups of the bead can be activated(e.g., via 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-Hydroxysuccinimide (NHS) or4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM)) such that they are reactive (e.g., reactive to amine functionalgroups where EDC/NHS or DMTMM are used for activation). The activatedCOOH groups can then react with an appropriate species (e.g., a speciescomprising an amine functional group where the carboxylic acid groupsare activated to be reactive with an amine functional group) comprisinga moiety to be linked to the bead.

In some embodiments, a bead can be formed from materials that includedegradable chemical cross-linkers, such as BAC or cystamine. Degradationof such degradable cross-linkers can be accomplished through a number ofmechanisms. In some examples, a bead can be contacted with a chemicaldegrading agent that can induce oxidation, reduction or other chemicalchanges. For example, a chemical degrading agent can be a reducingagent, such as dithiothreitol (DTT). Additional examples of reducingagents can include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane(dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), orcombinations thereof. A reducing agent can degrade the disulfide bondsformed between gel precursors forming the bead, and thus, degrade thebead.

A degradable bead can include one or more species with a labile bondsuch that, when the bead/species is exposed to the appropriate stimulus,the bond is broken and the bead degrades within the partition. Thelabile bond can be a chemical bond (e.g., covalent bond, ionic bond) orcan be another type of physical interaction (e.g., van der Waalsinteractions, dipole-dipole interactions, etc.). In some embodiments, across-linker used to generate a bead can include a labile bond. Uponexposure to the appropriate conditions, the labile bond can be brokenand the bead degraded. For example, a polyacrylamide bead featuringcystamine and linked, via a disulfide bond, to a barcode sequence, canbe combined with a reducing agent within a droplet of a water-in-oilemulsion. Within the droplet, the reducing agent can break the variousdisulfide bonds, resulting in bead degradation and release of thebarcode sequence into the aqueous, inner environment of the droplet. Inanother example, heating of a droplet with a bead-bound barcode sequencein basic solution can also result in bead degradation and release of theattached barcode sequence into the aqueous, inner environment of thedroplet. The free species (e.g., oligonucleotides, nucleic acidmolecules) can interact with other reagents contained in the partition.

A degradable bead can be useful in more quickly releasing an attachedspecies (e.g., a nucleic acid molecule, a barcode sequence, a primer,etc.) from the bead when the appropriate stimulus is applied to the beadas compared to a bead that does not degrade. For example, for a speciesbound to an inner surface of a porous bead or in the case of anencapsulated species, the species can have greater mobility andaccessibility to other species in solution upon degradation of the bead.In some embodiments, a species can also be attached to a degradable beadvia a degradable linker (e.g., disulfide linker). The degradable linkercan respond to the same stimuli as the degradable bead or the twodegradable species can respond to different stimuli. For example, abarcode sequence can be attached, via a disulfide bond, to apolyacrylamide bead comprising cystamine. Upon exposure of thebarcoded-bead to a reducing agent, the bead degrades and the barcodesequence is released upon breakage of both the disulfide linkage betweenthe barcode sequence and the bead and the disulfide linkages of thecystamine in the bead.

Any suitable number of species (e.g., primer, barcoded oligonucleotide)can be associated with a bead such that, upon release from the bead, thespecies (e.g., primer, e.g., barcoded oligonucleotide) are present inthe partition at a pre-defined concentration. Such pre-definedconcentration can be selected to facilitate certain reactions forgenerating a sequencing library, e.g., amplification, within thepartition. In some cases, the pre-defined concentration of the primercan be limited by the process of producing nucleic acid molecule (e.g.,oligonucleotide) bearing beads.

As will be appreciated from the above description, while referred to asdegradation of a bead, in many embodiments, degradation can refer to thedisassociation of a bound or entrained species from a bead, both withand without structurally degrading the physical bead itself. Forexample, entrained species can be released from beads through osmoticpressure differences due to, for example, changing chemicalenvironments. By way of example, alteration of bead pore volumes due toosmotic pressure differences can generally occur without structuraldegradation of the bead itself. In some cases, an increase in porevolume due to osmotic swelling of a bead can permit the release ofentrained species within the bead. In some embodiments, osmoticshrinking of a bead can cause a bead to better retain an entrainedspecies due to pore volume contraction.

Numerous chemical triggers can be used to trigger the degradation ofbeads within partitions. Examples of these chemical changes can include,but are not limited to pH-mediated changes to the integrity of acomponent within the bead, degradation of a component of a bead viacleavage of cross-linked bonds, and depolymerization of a component of abead.

In certain embodiments, a change in pH of a solution, such as anincrease in pH, can trigger degradation of a bead. In other embodiments,exposure to an aqueous solution, such as water, can trigger hydrolyticdegradation, and thus degradation of the bead. In some cases, anycombination of stimuli can trigger degradation of a bead. For example, achange in pH can enable a chemical agent (e.g., DTT) to become aneffective reducing agent.

Beads can also be induced to release their contents upon the applicationof a thermal stimulus. A change in temperature can cause a variety ofchanges to a bead. For example, heat can cause a solid bead to liquefy.A change in heat can cause melting of a bead such that a portion of thebead degrades. In other cases, heat can increase the internal pressureof the bead components such that the bead ruptures or explodes. Heat canalso act upon heat-sensitive polymers used as materials to constructbeads.

In addition to beads and analytes, partitions that are formed caninclude a variety of different reagents and species. For example, whenlysis reagents are present within the partitions, the lysis reagents canfacilitate the release of analytes within the partition. Examples oflysis agents include bioactive reagents, such as lysis enzymes that areused for lysis of different cell types, e.g., gram positive or negativebacteria, plants, yeast, mammalian, etc., such as lysozymes,achromopeptidase, lysostaphin, labiase, kitalase, lyticase, and avariety of other lysis enzymes available from, e.g., Sigma-Aldrich, Inc.(St. Louis, MO), as well as other commercially available lysis enzymes.Other lysis agents can additionally or alternatively be co-partitionedto cause the release analytes into the partitions. For example, in somecases, surfactant-based lysis solutions can be used to lyse cells,although these can be less desirable for emulsion based systems wherethe surfactants can interfere with stable emulsions. In someembodiments, lysis solutions can include non-ionic surfactants such as,for example, TritonX-100 and Tween 20. In some embodiments, lysissolutions can include ionic surfactants such as, for example, sarcosyland sodium dodecyl sulfate (SDS). Electroporation, thermal, acoustic ormechanical cellular disruption can also be used in certain embodiments,e.g., non-emulsion based partitioning such as encapsulation of analytesthat can be in addition to or in place of droplet partitioning, whereany pore size of the encapsulate is sufficiently small to retain nucleicacid fragments of a given volume, following cellular disruption.

Examples of other species that can be co-partitioned with analytes inthe partitions include, but are not limited to, DNase and RNaseinactivating agents or inhibitors, such as proteinase K, chelatingagents, such as EDTA, and other reagents employed in removing orotherwise reducing negative activity or impact of different cell lysatecomponents on subsequent processing of nucleic acids. Additionalreagents can also be co-partitioned, including endonucleases to fragmentDNA, DNA polymerase enzymes and dNTPs used to amplify nucleic acidfragments and to attach the barcode molecular tags to the amplifiedfragments.

Additional reagents can also include reverse transcriptase enzymes,including enzymes with terminal transferase activity, primers andoligonucleotides, and switch oligonucleotides (also referred to hereinas “switch oligos” or “template switching oligonucleotides”) which canbe used for template switching. In some embodiments, template switchingcan be used to increase the length of a cDNA. Template switching can beused to append a predefined nucleic acid sequence to the cDNA. In anexample of template switching, cDNA can be generated from reversetranscription of a template, e.g., cellular mRNA, where a reversetranscriptase with terminal transferase activity can add additionalnucleotides, e.g., poly(C), to the cDNA in a template independentmanner. Switch oligos can include sequences complementary to theadditional nucleotides, e.g., poly(G). The additional nucleotides (e.g.,poly(C)) on the cDNA can hybridize to the additional nucleotides (e.g.,poly(G)) on the switch oligo, whereby the switch oligo can be used bythe reverse transcriptase as template to further extend the cDNA.

Template switching oligonucleotides can include a hybridization regionand a template region. The hybridization region can include any sequencecapable of hybridizing to the target. In some cases, the hybridizationregion includes a series of G bases to complement the overhanging Cbases at the 3′ end of a cDNA molecule. The series of G bases caninclude 1 G base, 2 G bases, 3 G bases, 4 G bases, 5 G bases or morethan 5 G bases. The template sequence can include any sequence to beincorporated into the cDNA. In some cases, the template region includesat least 1 (e.g., at least 2, 3, 4, 5 or more) tag sequences and/orfunctional sequences. Switch oligos can include deoxyribonucleic acids;ribonucleic acids; bridged nucleic acids, modified nucleic acidsincluding 2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA), inverted dT,5-Methyl dC, 2′-deoxyInosine, Super T (5-hydroxybutynl-2′-deoxyuridine),Super G (8-aza-7-deazaguanosine), locked nucleic acids (LNAs), unlockednucleic acids (UNAs, e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC,2′ Fluoro bases (e.g., Fluoro C, Fluoro U, Fluoro A, and Fluoro G), andcombinations of the foregoing.

In some embodiments, beads that are partitioned with the analyte caninclude different types of oligonucleotides bound to the bead, where thedifferent types of oligonucleotides bind to different types of analytes.For example, a bead can include one or more first oligonucleotides(which can be capture probes, for example) that can bind or hybridize toa first type of analyte, such as mRNA for example, and one or moresecond oligonucleotides (which can be capture probes, for example) thatcan bind or hybridize to a second type of analyte, such as gDNA forexample. Partitions can also include lysis agents that aid in releasingnucleic acids from the co-partitioned cell, and can also include anagent (e.g., a reducing agent) that can degrade the bead and/or breakcovalent linkages between the oligonucleotides and the bead, releasingthe oligonucleotides into the partition. The released barcodedoligonucleotides (which can also be barcoded) can hybridize with mRNAreleased from the cell and also with gDNA released from the cell.

Barcoded constructs thus formed from hybridization can include a firsttype of construct that includes a sequence corresponding to an originalbarcode sequence from the bead and a sequence corresponding to atranscript from the cell, and a second type of construct that includes asequence corresponding to the original barcode sequence from the beadand a sequence corresponding to genomic DNA from the cell. The barcodedconstructs can then be released/removed from the partition and, in someembodiments, further processed to add any additional sequences. Theresulting constructs can then be sequenced, the sequencing dataprocessed, and the results used to spatially characterize the mRNA andthe gDNA from the cell.

In another example, a partition includes a bead that includes a firsttype of oligonucleotide (e.g., a first capture probe) with a firstbarcode sequence, a poly(T) priming sequence that can hybridize with thepoly(A) tail of an mRNA transcript, and a UMI barcode sequence that canuniquely identify a given transcript. The bead also includes a secondtype of oligonucleotide (e.g., a second capture probe) with a secondbarcode sequence, a targeted priming sequence that is capable ofspecifically hybridizing with a third barcoded oligonucleotide (e.g., ananalyte capture agent) coupled to an antibody that is bound to thesurface of the partitioned cell. The third barcoded oligonucleotideincludes a UMI barcode sequence that uniquely identifies the antibody(and thus, the particular cell surface feature to which it is bound).

In this example, the first and second barcoded oligonucleotides includethe same spatial barcode sequence (e.g., the first and second barcodesequences are the same), which permits downstream association ofbarcoded nucleic acids with the partition. In some embodiments, however,the first and second barcode sequences are different.

The partition also includes lysis agents that aid in releasing nucleicacids from the cell and can also include an agent (e.g., a reducingagent) that can degrade the bead and/or break a covalent linkage betweenthe barcoded oligonucleotides and the bead, releasing them into thepartition. The first type of released barcoded oligonucleotide canhybridize with mRNA released from the cell and the second type ofreleased barcoded oligonucleotide can hybridize with the third type ofbarcoded oligonucleotide, forming barcoded constructs.

The first type of barcoded construct includes a spatial barcode sequencecorresponding to the first barcode sequence from the bead and a sequencecorresponding to the UMI barcode sequence from the first type ofoligonucleotide, which identifies cell transcripts. The second type ofbarcoded construct includes a spatial barcode sequence corresponding tothe second barcode sequence from the second type of oligonucleotide, anda UMI barcode sequence corresponding to the third type ofoligonucleotide (e.g., the analyte capture agent) and used to identifythe cell surface feature. The barcoded constructs can then bereleased/removed from the partition and, in some embodiments, furtherprocessed to add any additional sequences. The resulting constructs arethen sequenced, sequencing data processed, and the results used tocharacterize the mRNA and cell surface feature of the cell.

The foregoing discussion involves two specific examples of beads witholigonucleotides for analyzing two different analytes within apartition. More generally, beads that are partitioned can have any ofthe structures described previously, and can include any of thedescribed combinations of oligonucleotides for analysis of two or more(e.g., three or more, four or more, five or more, six or more, eight ormore, ten or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 ormore, 40 or more, 50 or more) different types of analytes within apartition. Examples of beads with combinations of different types ofoligonucleotides (e.g., capture probes) for concurrently analyzingdifferent combinations of analytes within partitions include, but arenot limited to: (a) genomic DNA and cell surface features (e.g., usingthe analyte capture agents described herein); (b) mRNA and a lineagetracing construct; (c) mRNA and cell methylation status; (d) mRNA andaccessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq); (e)mRNA and cell surface or intracellular proteins and/or metabolites; (f)a barcoded analyte capture agent (e.g., the MHC multimers describedherein) and a V(D)J sequence of an immune cell receptor (e.g., T-cellreceptor); and (g) mRNA and a perturbation agent (e.g., a CRISPRcrRNA/sgRNA, TALEN, zinc finger nuclease, and/or antisenseoligonucleotide as described herein). In some embodiments, aperturbation agent can be a small molecule, an antibody, a drug, anaptamer, a miRNA, a physical environmental (e.g., temperature change),or any other known perturbation agents.

Additionally, in some embodiments, the unaggregated cell ordisaggregated cells introduced and processed within partitions ordroplets as described herein, can be removed from the partition,contacted with a spatial array, and spatially-barcoded according tomethods described herein. For example, single cells of an unaggregatedcell sample can be partitioned into partitions or droplets as describedherein. The partitions or droplets can include reagents to permeabilizea cell, barcode targeted cellular analyte(s) with a cellular barcode,and amplify the barcoded analytes. The partitions or droplets can becontacted with any of the spatial arrays described herein. In someembodiments, the partition can be dissolved, such that the contents ofthe partition are placed in contact with the capture probes of thespatial array. The capture probes of the spatial array can then capturetarget analytes from the ruptured partitions or the droplets, andprocessed by the spatial workflows described herein.

(G) Analysis of Captured Analytes (I) Sample Removal From an Array

In some embodiments, after contacting a biological sample with asubstrate that includes capture probes, a removal step can optionally beperformed to remove all or a portion of the biological sample from thesubstrate. In some embodiments, the removal step includes enzymaticand/or chemical degradation of cells of the biological sample. Forexample, the removal step can include treating the biological samplewith an enzyme (e.g., a proteinase, e.g., proteinase K) to remove atleast a portion of the biological sample from the substrate. In someembodiments, the removal step can include ablation of the tissue (e.g.,laser ablation).

In some embodiments, provided herein are methods for spatially detectingan analyte (e.g., detecting the location of an analyte, e.g., abiological analyte) from a biological sample (e.g., present in abiological sample), the method comprising: (a) optionally stainingand/or imaging a biological sample on a substrate; (b) permeabilizing(e.g., providing a solution comprising a permeabilization reagent to)the biological sample on the substrate; (c) contacting the biologicalsample with an array comprising a plurality of capture probes, wherein acapture probe of the plurality captures the biological analyte; and (d)analyzing the captured biological analyte, thereby spatially detectingthe biological analyte; wherein the biological sample is fully orpartially removed from the substrate.

In some embodiments, a biological sample is not removed from thesubstrate. For example, the biological sample is not removed from thesubstrate prior to releasing a capture probe (e.g., a capture probebound to an analyte) from the substrate. In some embodiments, suchreleasing comprises cleavage of the capture probe from the substrate(e.g., via a cleavage domain). In some embodiments, such releasing doesnot comprise releasing the capture probe from the substrate (e.g., acopy of the capture probe bound to an analyte can be made and the copycan be released from the substrate, e.g., via denaturation). In someembodiments, the biological sample is not removed from the substrateprior to analysis of an analyte bound to a capture probe after it isreleased from the substrate. In some embodiments, the biological sampleremains on the substrate during removal of a capture probe from thesubstrate and/or analysis of an analyte bound to the capture probe afterit is released from the substrate. In some embodiments, analysis of ananalyte bound to capture probe from the substrate can be performedwithout subjecting the biological sample to enzymatic and/or chemicaldegradation of the cells (e.g., permeabilized cells) or ablation of thetissue (e.g., laser ablation).

In some embodiments, at least a portion of the biological sample is notremoved from the substrate. For example, a portion of the biologicalsample can remain on the substrate prior to releasing a capture probe(e.g., a capture prove bound to an analyte) from the substrate and/oranalyzing an analyte bound to a capture probe released from thesubstrate. In some embodiments, at least a portion of the biologicalsample is not subjected to enzymatic and/or chemical degradation of thecells (e.g., permeabilized cells) or ablation of the tissue (e.g., laserablation) prior to analysis of an analyte bound to a capture probe fromthe substrate.

In some embodiments, provided herein are methods for spatially detectingan analyte (e.g., detecting the location of an analyte, e.g., abiological analyte) from a biological sample (e.g., present in abiological sample) that include: (a) optionally staining and/or imaginga biological sample on a substrate; (b) permeabilizing (e.g., providinga solution comprising a permeabilization reagent to) the biologicalsample on the substrate; (c) contacting the biological sample with anarray comprising a plurality of capture probes, wherein a capture probeof the plurality captures the biological analyte; and (d) analyzing thecaptured biological analyte, thereby spatially detecting the biologicalanalyte; where the biological sample is not removed from the substrate.

In some embodiments, provided herein are methods for spatially detectinga biological analyte of interest from a biological sample that include:(a) staining and imaging a biological sample on a substrate; (b)providing a solution comprising a permeabilization reagent to thebiological sample on the substrate; (c) contacting the biological samplewith an array on a substrate, wherein the array comprises one or morecapture probe pluralities thereby allowing the one or more pluralitiesof capture probes to capture the biological analyte of interest; and (d)analyzing the captured biological analyte, thereby spatially detectingthe biological analyte of interest; where the biological sample is notremoved from the substrate.

In some embodiments, the method further includes selecting a region ofinterest in the biological sample to subject to spatial transcriptomicanalysis. In some embodiments, one or more of the one or more captureprobes include a capture domain. In some embodiments, one or more of theone or more capture probe pluralities comprise a unique molecularidentifier (UMI). In some embodiments, one or more of the one or morecapture probe pluralities comprise a cleavage domain. In someembodiments, the cleavage domain comprises a sequence recognized andcleaved by a uracil-DNA glycosylase, apurinic/apyrimidinic (AP)endonuclease (APE1), U uracil-specific excision reagent (USER), and/oran endonuclease VIII. In some embodiments, one or more capture probes donot comprise a cleavage domain and is not cleaved from the array.

(II) Extended Capture Probes

In some embodiments, a capture probe can be extended (an “extendedcapture probe,” e.g., as described herein (e.g., Section II(b)(vii))).For example, extending a capture probe can include generating cDNA froma captured (hybridized) RNA. This process involves synthesis of acomplementary strand of the hybridized nucleic acid, e.g., generatingcDNA based on the captured RNA template (the RNA hybridized to thecapture domain of the capture probe). Thus, in an initial step ofextending a capture probe, e.g., the cDNA generation, the captured(hybridized) nucleic acid, e.g., RNA, acts as a template for theextension, e.g., reverse transcription, step.

In some embodiments, the capture probe is extended using reversetranscription. For example, reverse transcription includes synthesizingcDNA (complementary or copy DNA) from RNA, e.g., (messenger RNA), usinga reverse transcriptase. In some embodiments, reverse transcription isperformed while the tissue is still in place, generating an analytelibrary, where the analyte library includes the spatial barcodes fromthe adjacent capture probes. In some embodiments, the capture probe isextended using one or more DNA polymerases.

In some embodiments, a capture domain of a capture probe includes aprimer for producing the complementary strand of a nucleic acidhybridized to the capture probe, e.g., a primer for DNA polymeraseand/or reverse transcription. The nucleic acid, e.g., DNA and/or cDNA,molecules generated by the extension reaction incorporate the sequenceof the capture probe. The extension of the capture probe, e.g., a DNApolymerase and/or reverse transcription reaction, can be performed usinga variety of suitable enzymes and protocols.

In some embodiments, a full-length DNA (e.g., cDNA) molecule isgenerated. In some embodiments, a “full-length” DNA molecule refers tothe whole of the captured nucleic acid molecule. However, if a nucleicacid (e.g., RNA) was partially degraded in the tissue sample, then thecaptured nucleic acid molecules will not be the same length as theinitial RNA in the tissue sample. In some embodiments, the 3′ end of theextended probes, e.g., first strand cDNA molecules, is modified. Forexample, a linker or adaptor can be ligated to the 3′ end of theextended probes. This can be achieved using single stranded ligationenzymes such as T4 RNA ligase or Circligase™ (available from EpicentreBiotechnologies, Madison, WI). In some embodiments, template switchingoligonucleotides are used to extend cDNA in order to generate afull-length cDNA (or as close to a full-length cDNA as possible). Insome embodiments, a second strand synthesis helper probe (a partiallydouble stranded DNA molecule capable of hybridizing to the 3′ end of theextended capture probe), can be ligated to the 3′ end of the extendedprobe, e.g., first strand cDNA, molecule using a double strandedligation enzyme such as T4 DNA ligase. Other enzymes appropriate for theligation step are known in the art and include, e.g., Tth DNA ligase,Taq DNA ligase, Thermococcus sp. (strain 9°N) DNA ligase (9°N™ DNAligase, New England Biolabs), Ampligase™ (available from EpicentreBiotechnologies, Madison, WI), and SplintR (available from New EnglandBiolabs, Ipswich, MA). In some embodiments, a polynucleotide tail, e.g.,a poly(A) tail, is incorporated at the 3′ end of the extended probemolecules. In some embodiments, the polynucleotide tail is incorporatedusing a terminal transferase active enzyme.

In some embodiments, double-stranded extended capture probes are treatedto remove any unextended capture probes prior to amplification and/oranalysis, e.g., sequence analysis. This can be achieved by a variety ofmethods, e.g., using an enzyme to degrade the unextended probes, such asan exonuclease enzyme, or purification columns.

In some embodiments, extended capture probes are amplified to yieldquantities that are sufficient for analysis, e.g., via DNA sequencing.In some embodiments, the first strand of the extended capture probes(e.g., DNA and/or cDNA molecules) acts as a template for theamplification reaction (e.g., a polymerase chain reaction).

In some embodiments, the amplification reaction incorporates an affinitygroup onto the extended capture probe (e.g., RNA-cDNA hybrid) using aprimer including the affinity group. In some embodiments, the primerincludes an affinity group and the extended capture probes includes theaffinity group. The affinity group can correspond to any of the affinitygroups described previously.

In some embodiments, the extended capture probes including the affinitygroup can be coupled to a substrate specific for the affinity group. Insome embodiments, the substrate can include an antibody or antibodyfragment. In some embodiments, the substrate includes avidin orstreptavidin and the affinity group includes biotin. In someembodiments, the substrate includes maltose and the affinity groupincludes maltose-binding protein. In some embodiments, the substrateincludes maltose-binding protein and the affinity group includesmaltose. In some embodiments, amplifying the extended capture probes canfunction to release the extended probes from the surface of thesubstrate, insofar as copies of the extended probes are not immobilizedon the substrate.

In some embodiments, the extended capture probe or complement oramplicon thereof is released. The step of releasing the extended captureprobe or complement or amplicon thereof from the surface of thesubstrate can be achieved in a number of ways. In some embodiments, anextended capture probe or a complement thereof is released from thearray by nucleic acid cleavage and/or by denaturation (e.g., by heatingto denature a double-stranded molecule).

In some embodiments, the extended capture probe or complement oramplicon thereof is released from the surface of the substrate (e.g.,array) by physical means. For example, where the extended capture probeis indirectly immobilized on the array substrate, e.g., viahybridization to a surface probe, it can be sufficient to disrupt theinteraction between the extended capture probe and the surface probe.Methods for disrupting the interaction between nucleic acid moleculesinclude denaturing double stranded nucleic acid molecules are known inthe art. A straightforward method for releasing the DNA molecules (i.e.,of stripping the array of extended probes) is to use a solution thatinterferes with the hydrogen bonds of the double stranded molecules. Insome embodiments, the extended capture probe is released by a applyingheated solution, such as water or buffer, of at least 85° C., e.g., atleast 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99° C. In some embodiments,a solution including salts, surfactants, etc. that can furtherdestabilize the interaction between the nucleic acid molecules is addedto release the extended capture probe from the substrate.

In some embodiments, where the extended capture probe includes acleavage domain, the extended capture probe is released from the surfaceof the substrate by cleavage. For example, the cleavage domain of theextended capture probe can be cleaved by any of the methods describedherein. In some embodiments, the extended capture probe is released fromthe surface of the substrate, e.g., via cleavage of a cleavage domain inthe extended capture probe, prior to the step of amplifying the extendedcapture probe.

(III) Cleavage Domain

Capture probes can optionally include a “cleavage domain,” where one ormore segments or regions of the capture probe (e.g., spatial barcodesand/or UMIs) can be releasably, cleavably, or reversibly attached to afeature, or some other substrate, so that spatial barcodes and/or UMIscan be released or be releasable through cleavage of a linkage betweenthe capture probe and the feature, or released through degradation ofthe underlying substrate or chemical substrate, allowing the spatialbarcode(s) and/or UMI(s) of the cleaved capture probe to be accessed orbe accessible by other reagents, or both. Non-limiting aspects ofcleavage domains are described herein (e.g., in Section II(b)(ii)).

In some embodiments, the capture probe is linked, (e.g., via a disulfidebond), to a feature. In some embodiments, the capture probe is linked toa feature via a propylene group (e.g., Spacer C3). A reducing agent canbe added to break the various disulfide bonds, resulting in release ofthe capture probe including the spatial barcode sequence. In anotherexample, heating can also result in degradation and release of theattached capture probe. In some embodiments, the heating is done bylaser (e.g., laser ablation) and features at specific locations can bedegraded. In addition to thermally cleavable bonds, disulfide bonds,photosensitive bonds, and UV sensitive bonds, other non-limitingexamples of labile bonds that can be coupled to a capture probe (e.g.,spatial barcode) include an ester linkage (e.g., cleavable with an acid,a base, or hydroxylamine), a vicinal diol linkage (e.g., cleavable viasodium periodate), a Diels-Alder linkage (e.g., cleavable via heat), asulfone linkage (e.g., cleavable via a base), a silyl ether linkage(e.g., cleavable via an acid), a glycosidic linkage (e.g., cleavable viaan amylase), a peptide linkage (e.g., cleavable via a protease), or aphosphodiester linkage (e.g., cleavable via a nuclease (e.g., DNAase)).

In some embodiments, the cleavage domain includes a poly(U) sequencewhich can be cleaved by a mixture of Uracil DNA glycosylase (UDG) andthe DNA glycosylase-lyase Endonuclease VIII, commercially known as theUSER™ enzyme. In some embodiments, the cleavage domain can be a singleU. In some embodiments, the cleavage domain can be an abasic site thatcan be cleaved with an abasic site-specific endonuclease (e.g.,Endonuclease IV or Endonuclease VIII).

In some embodiments, the cleavage domain of the capture probe is anucleotide sequence within the capture probe that is cleavedspecifically, e.g., physically by light or heat, chemically orenzymatically. The location of the cleavage domain within the captureprobe will depend on whether or not the capture probe is immobilized onthe substrate such that it has a free 3′ end capable of functioning asan extension primer (e.g., by its 5′ or 3′ end). For example, if thecapture probe is immobilized by its 5′ end, the cleavage domain will belocated 5′ to the spatial barcode and/or UMI, and cleavage of saiddomain results in the release of part of the capture probe including thespatial barcode and/or UMI and the sequence 3′ to the spatial barcode,and optionally part of the cleavage domain, from a feature.Alternatively, if the capture probe is immobilized by its 3′ end, thecleavage domain will be located 3′ to the capture domain (and spatialbarcode) and cleavage of said domain results in the release of part ofthe capture probe including the spatial barcode and the sequence 3′ tothe spatial barcode from a feature. In some embodiments, cleavageresults in partial removal of the cleavage domain. In some embodiments,cleavage results in complete removal of the cleavage domain,particularly when the capture probes are immobilized via their 3′ end asthe presence of a part of the cleavage domain can interfere with thehybridization of the capture domain and the target nucleic acid and/orits subsequent extension.

(IV) Sequencing

After analytes from the sample have hybridized or otherwise beenassociated with capture probes, analyte capture agents, or otherbarcoded oligonucleotide sequences according to any of the methodsdescribed above in connection with the general spatial cell-basedanalytical methodology, the barcoded constructs that result fromhybridization/association are analyzed via sequencing to identify theanalytes.

In some embodiments, where a sample is barcoded directly viahybridization with capture probes or analyte capture agents hybridized,bound, or associated with either the cell surface, or introduced intothe cell, as described above, sequencing can be performed on the intactsample. Alternatively, if the barcoded sample has been separated intofragments, cell groups, or individual cells, as described above,sequencing can be performed on individual fragments, cell groups, orcells. For analytes that have been barcoded via partitioning with beads,as described above, individual analytes (e.g., cells, or cellularcontents following lysis of cells) can be extracted from the partitionsby breaking the partitions, and then analyzed by sequencing to identifythe analytes.

A wide variety of different sequencing methods can be used to analyzebarcoded analyte constructs. In general, sequenced polynucleotides canbe, for example, nucleic acid molecules such as deoxyribonucleic acid(DNA) or ribonucleic acid (RNA), including variants or derivativesthereof (e.g., single stranded DNA or DNA/RNA hybrids, and nucleic acidmolecules with a nucleotide analog).

Sequencing of polynucleotides can be performed by various commercialsystems. More generally, sequencing can be performed using nucleic acidamplification, polymerase chain reaction (PCR) (e.g., digital PCR anddroplet digital PCR (ddPCR), quantitative PCR, real time PCR, multiplexPCR, PCR-based singleplex methods, emulsion PCR), and/or isothermalamplification.

Other examples of methods for sequencing genetic material include, butare not limited to, DNA hybridization methods (e.g., Southern blotting),restriction enzyme digestion methods, Sanger sequencing methods,next-generation sequencing methods (e.g., single-molecule real-timesequencing, nanopore sequencing, and Polony sequencing), ligationmethods, and microarray methods. Additional examples of sequencingmethods that can be used include targeted sequencing, single moleculereal-time sequencing, exon sequencing, electron microscopy-basedsequencing, panel sequencing, transistor-mediated sequencing, directsequencing, random shotgun sequencing, Sanger dideoxy terminationsequencing, whole-genome sequencing, sequencing by hybridization,pyrosequencing, capillary electrophoresis, gel electrophoresis, duplexsequencing, cycle sequencing, single-base extension sequencing,solid-phase sequencing, high-throughput sequencing, massively parallelsignature sequencing, co-amplification at lower denaturationtemperature-PCR (COLD-PCR), sequencing by reversible dye terminator,paired-end sequencing, near-term sequencing, exonuclease sequencing,sequencing by ligation, short-read sequencing, single-moleculesequencing, sequencing-by-synthesis, real-time sequencing,reverse-terminator sequencing, nanopore sequencing, 454 sequencing,Solexa Genome Analyzer sequencing, SOLiD™ sequencing, MS-PET sequencing,and any combinations thereof.

Sequence analysis of the nucleic acid molecules (including barcodednucleic acid molecules or derivatives thereof) can be direct orindirect. Thus, the sequence analysis substrate (which can be viewed asthe molecule which is subjected to the sequence analysis step orprocess) can be the barcoded nucleic acid molecule or it can be amolecule which is derived therefrom (e.g., a complement thereof). Thus,for example, in the sequence analysis step of a sequencing reaction, thesequencing template can be the barcoded nucleic acid molecule or it canbe a molecule derived therefrom. For example, a first and/or secondstrand DNA molecule can be directly subjected to sequence analysis(e.g., sequencing), i.e., can directly take part in the sequenceanalysis reaction or process (e.g., the sequencing reaction orsequencing process, or be the molecule which is sequenced or otherwiseidentified). Alternatively, the barcoded nucleic acid molecule can besubjected to a step of second strand synthesis or amplification beforesequence analysis (e.g., sequencing or identification by anothertechnique). The sequence analysis substrate (e.g., template) can thus bean amplicon or a second strand of a barcoded nucleic acid molecule.

In some embodiments, both strands of a double stranded molecule can besubjected to sequence analysis (e.g., sequenced). In some embodiments,single stranded molecules (e.g., barcoded nucleic acid molecules) can beanalyzed (e.g., sequenced). To perform single molecule sequencing, thenucleic acid strand can be modified at the 3′ end.

Massively parallel pyrosequencing techniques can be used for sequencingnucleic acids. In pyrosequencing, the nucleic acid is amplified insidewater droplets in an oil solution (emulsion PCR), with each dropletcontaining a single nucleic acid template attached to a singleprimer-coated bead that then forms a clonal colony. The sequencingsystem contains many picolitre-volume wells each containing a singlebead and sequencing enzymes. Pyrosequencing uses luciferase to generatelight for detection of the individual nucleotides added to the nascentnucleic acid and the combined data are used to generate sequence reads.

As another example application of pyrosequencing, released PPi can bedetected by being immediately converted to adenosine triphosphate (ATP)by ATP sulfurylase, and the level of ATP generated can be detected vialuciferase-produced photons, such as described in Ronaghi, et al., Anal.Biochem. 242(1), 84-9 (1996); Ronaghi, Genome Res. 11(1), 3-11 (2001);Ronaghi et al. Science 281 (5375), 363 (1998); and U.S. Pat. Nos.6,210,891, 6,258,568, and 6,274,320, the entire contents of each ofwhich are incorporated herein by reference.

In some embodiments, a massively parallel sequencing technique can bebased on reversible dye-terminators. As an example, DNA molecules arefirst attached to primers on, e.g., a glass or silicon substrate, andamplified so that local clonal colonies are formed (bridgeamplification). Four types of ddNTPs are added, and non-incorporatednucleotides are washed away. Unlike pyrosequencing, the DNA is onlyextended one nucleotide at a time due to a blocking group (e.g., 3′blocking group present on the sugar moiety of the ddNTP). A detectoracquires images of the fluorescently labelled nucleotides, and then thedye along with the terminal 3′ blocking group is chemically removed fromthe DNA, as a precursor to a subsequent cycle. This process can berepeated until the required sequence data is obtained.

In some embodiments, sequencing is performed by detection of hydrogenions that are released during the polymerization of DNA. A microwellcontaining a template DNA strand to be sequenced can be flooded with asingle type of nucleotide. If the introduced nucleotide is complementaryto the leading template nucleotide, it is incorporated into the growingcomplementary strand. This causes the release of a hydrogen ion thattriggers a hypersensitive ion sensor, which indicates that a reactionhas occurred. If homopolymer repeats are present in the templatesequence, multiple nucleotides will be incorporated in a single cycle.This leads to a corresponding number of released hydrogen ions and aproportionally higher electronic signal.

In some embodiments, sequencing can be performed in situ. In situsequencing methods are particularly useful, for example, when thebiological sample remains intact after analytes on the sample surface(e.g., cell surface analytes) or within the sample (e.g., intracellularanalytes) have been barcoded. In situ sequencing typically involvesincorporation of a labeled nucleotide (e.g., fluorescently labeledmononucleotides or dinucleotides) in a sequential, template-dependentmanner or hybridization of a labeled primer (e.g., a labeled randomhexamer) to a nucleic acid template such that the identities (i.e.,nucleotide sequence) of the incorporated nucleotides or labeled primerextension products can be determined, and consequently, the nucleotidesequence of the corresponding template nucleic acid. Aspects of in situsequencing are described, for example, in Mitra et al., (2003) Anal.Biochem. 320, 55-65, and Lee et al., (2014) Science, 343(6177),1360-1363, the entire contents of each of which are incorporated hereinby reference.

In addition, examples of methods and systems for performing in situsequencing are described in PCT Pat. Application Publication Nos.WO2014/163886, WO2018/045181, WO2018/045186, and in U.S. Pat. Nos.10,138,509 and 10,179,932, the entire contents of each of which areincorporated herein by reference. Exemplary techniques for in situsequencing include, but are not limited to, STARmap (described forexample in Wang et al., (2018) Science, 361(6499) 5691), MERFISH(described for example in Moffitt, (2016) Methods in Enzymology, 572,1-49), and FISSEQ (described for example in U.S. Pat. ApplicationPublication No. 2019/0032121). The entire contents of each of theforegoing references are incorporated herein by reference.

For analytes that have been barcoded via partitioning, barcoded nucleicacid molecules or derivatives thereof (e.g., barcoded nucleic acidmolecules to which one or more functional sequences have been added, orfrom which one or more features have been removed) can be pooled andprocessed together for subsequent analysis such as sequencing on highthroughput sequencers. Processing with pooling can be implemented usingbarcode sequences. For example, barcoded nucleic acid molecules of agiven partition can have the same barcode, which is different frombarcodes of other spatial partitions. Alternatively, barcoded nucleicacid molecules of different partitions can be processed separately forsubsequent analysis (e.g., sequencing).

In some embodiments, where capture probes do not contain a spatialbarcode, the spatial barcode can be added after the capture probecaptures analytes from a biological sample and before analysis of theanalytes. When a spatial barcode is added after an analyte is captured,the barcode can be added after amplification of the analyte (e.g.,reverse transcription and polymerase amplification of RNA). In someembodiments, analyte analysis uses direct sequencing of one or morecaptured analytes, such as direct sequencing of hybridized RNA. In someembodiments, direct sequencing is performed after reverse transcriptionof hybridized RNA. In some embodiments direct sequencing is performedafter amplification of reverse transcription of hybridized RNA.

In some embodiments, direct sequencing of captured RNA is performed bysequencing-by-synthesis (SBS). In some embodiments, a sequencing primeris complementary to a sequence in one or more of the domains of acapture probe (e.g., functional domain). In such embodiments,sequencing-by-synthesis can include reverse transcription and/oramplification in order to generate a template sequence (e.g., functionaldomain) from which a primer sequence can bind.

SBS can involve hybridizing an appropriate primer, sometimes referred toas a sequencing primer, with the nucleic acid template to be sequenced,extending the primer, and detecting the nucleotides used to extend theprimer. Preferably, the nucleic acid used to extend the primer isdetected before a further nucleotide is added to the growing nucleicacid chain, thus allowing base-by-base in situ nucleic acid sequencing.The detection of incorporated nucleotides is facilitated by includingone or more labelled nucleotides in the primer extension reaction. Toallow the hybridization of an appropriate sequencing primer to thenucleic acid template to be sequenced, the nucleic acid template shouldnormally be in a single stranded form. If the nucleic acid templatesmaking up the nucleic acid features are present in a double strandedform these can be processed to provide single stranded nucleic acidtemplates using methods well known in the art, for example bydenaturation, cleavage, etc. The sequencing primers which are hybridizedto the nucleic acid template and used for primer extension arepreferably short oligonucleotides, for example, 15 to 25 nucleotides inlength. The sequencing primers can be provided in solution or in animmobilized form. Once the sequencing primer has been annealed to thenucleic acid template to be sequenced by subjecting the nucleic acidtemplate and sequencing primer to appropriate conditions, primerextension is carried out, for example using a nucleic acid polymeraseand a supply of nucleotides, at least some of which are provided in alabelled form, and conditions suitable for primer extension if asuitable nucleotide is provided.

Preferably after each primer extension step, a washing step is includedin order to remove unincorporated nucleotides which can interfere withsubsequent steps. Once the primer extension step has been carried out,the nucleic acid colony is monitored to determine whether a labellednucleotide has been incorporated into an extended primer. The primerextension step can then be repeated to determine the next and subsequentnucleotides incorporated into an extended primer. If the sequence beingdetermined is unknown, the nucleotides applied to a given colony areusually applied in a chosen order which is then repeated throughout theanalysis, for example dATP, dTTP, dCTP, dGTP.

SBS techniques which can be used are described for example, but notlimited to, those in U.S. Pat. App. Pub. No. 2007/0166705, U.S. Pat.App. Pub. No. 2006/0188901, U.S. Pat. 7,057,026, U.S. Pat. App. Pub. No.2006/0240439, U.S. Pat. App. Pub. No. 2006/0281109, PCT Pat. App. Pub.No. WO 05/065814, U.S. Pat. App. Pub. No. 2005/0100900, PCT Pat. App.Pub. No. WO 06/064199, PCT Pat. App. Pub. No. WO07/010,251, U.S. Pat.App. Pub. No. 2012/0270305, U.S. Pat. App. Pub. No. 2013/0260372, andU.S. Pat. App. Pub. No. 2013/0079232, the entire contents of each ofwhich are incorporated herein by reference.

In some embodiments, direct sequencing of captured RNA is performed bysequential fluorescence hybridization (e.g., sequencing byhybridization). In some embodiments, a hybridization reaction where RNAis hybridized to a capture probe is performed in situ. In someembodiments, captured RNA is not amplified prior to hybridization with asequencing probe. In some embodiments, RNA is amplified prior tohybridization with sequencing probes (e.g., reverse transcription tocDNA and amplification of cDNA). In some embodiments, amplification isperformed using single-molecule hybridization chain reaction. In someembodiments, amplification is performed using rolling chainamplification.

Sequential fluorescence hybridization can involve sequentialhybridization of probes including degenerate primer sequences and adetectable label. A degenerate primer sequence is a shortoligonucleotide sequence which is capable of hybridizing to any nucleicacid fragment independent of the sequence of said nucleic acid fragment.For example, such a method could include the steps of: (a) providing amixture including four probes, each of which includes either A, C, G, orT at the 5′-terminus, further including degenerate nucleotide sequenceof 5 to 11 nucleotides in length, and further including a functionaldomain (e.g., fluorescent molecule) that is distinct for probes with A,C, G, or T at the 5′-terminus; (b) associating the probes of step (a) tothe target polynucleotide sequences, whose sequence needs will bedetermined by this method; (c) measuring the activities of the fourfunctional domains and recording the relative spatial location of theactivities; (d) removing the reagents from steps (a)-(b) from the targetpolynucleotide sequences; and repeating steps (a)-(d) for n cycles,until the nucleotide sequence of the spatial domain for each bead isdetermined, with modification that the oligonucleotides used in step (a)are complementary to part of the target polynucleotide sequences and thepositions 1 through n flanking the part of the sequences. Because thebarcode sequences are different, in some embodiments, these additionalflanking sequences are degenerate sequences. The fluorescent signal fromeach spot on the array for cycles 1 through n can be used to determinethe sequence of the target polynucleotide sequences.

In some embodiments, direct sequencing of captured RNA using sequentialfluorescence hybridization is performed in vitro. In some embodiments,captured RNA is amplified prior to hybridization with a sequencing probe(e.g., reverse transcription to cDNA and amplification of cDNA). In someembodiments, a capture probe containing captured RNA is exposed to thesequencing probe targeting coding regions of RNA. In some embodiments,one or more sequencing probes are targeted to each coding region. Insome embodiments, the sequencing probe is designed to hybridize withsequencing reagents (e.g., a dye-labeled readout oligonucleotides). Asequencing probe can then hybridize with sequencing reagents. In someembodiments, output from the sequencing reaction is imaged. In someembodiments, a specific sequence of cDNA is resolved from an image of asequencing reaction. In some embodiments, reverse transcription ofcaptured RNA is performed prior to hybridization to the sequencingprobe. In some embodiments, the sequencing probe is designed to targetcomplementary sequences of the coding regions of RNA (e.g., targetingcDNA).

In some embodiments, a captured RNA is directly sequenced using ananopore-based method. In some embodiments, direct sequencing isperformed using nanopore direct RNA sequencing in which captured RNA istranslocated through a nanopore. A nanopore current can be recorded andconverted into a base sequence. In some embodiments, captured RNAremains attached to a substrate during nanopore sequencing. In someembodiments, captured RNA is released from the substrate prior tonanopore sequencing. In some embodiments, where the analyte of interestis a protein, direct sequencing of the protein can be performed usingnanopore-based methods. Examples of nanopore-based sequencing methodsthat can be used are described in Deamer et al., Trends Biotechnol. 18,14 7-151 (2000); Deamer et al., Acc. Chem. Res. 35:817-825 (2002); Li etal., Nat. Mater. 2:611-615 (2003); Soni et al., Clin. Chem. 53,1996-2001 (2007); Healy et al., Nanomed. 2, 459-481 (2007); Cockroft etal., J. Am. Chem. Soc. 130, 818-820 (2008); and in U.S. Pat. 7,001,792.The entire contents of each of the foregoing references are incorporatedherein by reference.

In some embodiments, direct sequencing of captured RNA is performedusing single molecule sequencing by ligation. Such techniques utilizeDNA ligase to incorporate oligonucleotides and identify theincorporation of such oligonucleotides. The oligonucleotides typicallyhave different labels that are correlated with the identity of aparticular nucleotide in a sequence to which the oligonucleotideshybridize. Aspects and features involved in sequencing by ligation aredescribed, for example, in Shendure et al. Science (2005), 309:1728-1732, and in U.S. Pat. Nos. 5,599,675; 5,750,341; 6,969,488;6,172,218; and 6,306,597, the entire contents of each of which areincorporated herein by reference.

In some embodiments, nucleic acid hybridization can be used forsequencing. These methods utilize labeled nucleic acid decoder probesthat are complementary to at least a portion of a barcode sequence.Multiplex decoding can be performed with pools of many different probeswith distinguishable labels. Non-limiting examples of nucleic acidhybridization sequencing are described for example in U.S. Pat. No.8,460,865, and in Gunderson et al., Genome Research 14:870-877 (2004),the entire contents of each of which are incorporated herein byreference.

In some embodiments, commercial high-throughput digital sequencingtechniques can be used to analyze barcode sequences, in which DNAtemplates are prepared for sequencing not one at a time, but in a bulkprocess, and where many sequences are read out preferably in parallel,or alternatively using an ultra-high throughput serial process thatitself may be parallelized. Examples of such techniques includeIllumina^(®) sequencing (e.g., flow cell-based sequencing techniques),sequencing by synthesis using modified nucleotides (such ascommercialized in TruSeq™ and HiSeq™ technology by Illumina, Inc., SanDiego, CA), HeliScope™ by Helicos Biosciences Corporation, Cambridge,MA, and PacBio RS by Pacific Biosciences of California, Inc., MenloPark, CA), sequencing by ion detection technologies (Ion Torrent, Inc.,South San Francisco, CA), and sequencing of DNA nanoballs (CompleteGenomics, Inc., Mountain View, CA).

In some embodiments, detection of a proton released upon incorporationof a nucleotide into an extension product can be used in the methodsdescribed herein. For example, the sequencing methods and systemsdescribed in U.S. Patent Application Publication Nos. 2009/0026082,2009/0127589, 2010/0137143, and 2010/0282617, can be used to directlysequence barcodes.

In some embodiments, real-time monitoring of DNA polymerase activity canbe used during sequencing. For example, nucleotide incorporations can bedetected through fluorescence resonance energy transfer (FRET), asdescribed for example in Levene et al., Science (2003), 299, 682-686,Lundquist et al., Opt. Lett. (2008), 33, 1026-1028, and Korlach et al.,Proc. Natl. Acad. Sci. USA (2008), 105, 1176-1181. The entire contentsof each of the foregoing references are incorporated herein by referenceherein.

(V) Temporal Analysis

In some embodiments, the methods described herein can be used to assessanalyte levels and/or expression in a cell or a biological sample overtime (e.g., before or after treatment with an agent or different stagesof differentiation). In some examples, the methods described herein canbe performed on multiple similar biological samples or cells obtainedfrom the subject at a different time points (e.g., before or aftertreatment with an agent, different stages of differentiation, differentstages of disease progression, different ages of the subject, before orafter physical perturbation, before or after treatment with aperturbation agent as described herein, or before or after developmentof resistance to an agent). As described herein, a “perturbation agent”or “perturbation reagent” can be a small molecule, an antibody, a drug,an aptamer, a nucleic acid (e.g., miRNA), a CRISPR crRNA/sgRNA, TALEN,zinc finger nuclease, antisense oligonucleotide a physical environmental(e.g., temperature change), and/or any other known perturbation agentswhere the agent alters equilibrium or homeostasis.

In some embodiments, the methods described herein can be performed onmultiple similar biological samples or cells obtained from the subjectat 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. For example, the multiplesimilar biological samples can be repetitive samples from the samesubject, the same tissue, the same organoid, the same cell suspension,or any other biological sample described herein. In some embodiments,the methods described herein can be performed on the same biologicalsample or cells obtained from the subject at a different time points(e.g., before or after treatment with a perturbation agent, differentstages of differentiation, different stages of disease progression,different ages of the subject, or before or after development ofresistance to an agent). In some embodiments, a perturbation agent canbe small-molecules, antibodies, nucleic acids, peptides, and/or otherexternal stimuli (e.g., temperature change). In some embodiments, thebiological sample is contacted with a different array at each timepoint.

In some embodiments, a sample can be placed in a controlled environmentpermissive for cellular growth and/or maintenance, and/or to preventhypoxia. In some embodiments, a controlled environment allows a sampleto be analyzed at different time points. Barcoded arrays can be placedproximal to (e.g., on top of) the sample and imaged using a microscopeor other suitable instrument to register the relative position of thebiological sample to the barcoded array, optionally using opticallyencoded fiducial markers. An electric field can be applied for a periodof time, such that biological analytes (e.g., DNA, RNA, proteins,metabolites, small molecules, lipids, and the like) are released fromthe sample and captured by capture probes on the spatially-barcodedarray, preserving spatial information of the sample. The barcoded arraycan be removed, and the spatial and molecular information therein isdetermined (e.g., by performing library construction for next generationsequencing or in situ sequencing). Sequencing can be followed bycomputational analysis to correlate the molecular information (e.g.,gene expression values with the spatial barcode). These steps can berepeated one or more times to capture the spatial information ofanalytes at different time-points.

In some embodiments, methods as described herein can be combined with acell migration assay. A cell migration assay can comprise one or moremicroprinted lines, or suspended 3D nanofibers, on which the cellsmigrate. Migration using these assays can be measured by imaging cellmigration and/or contacting migrated cells with a spatially-barcodedarray. An array used in a cell migration assay can comprise one or morechannels on the substrate of the array, e.g., to confine cell migrationto one dimension along the substrate. Additionally, the channels candirect the migration of a cell such that it does not contact anothercell on the array (e.g., the channels do not overlap with each other),and in some embodiments, the channels are about the same width as orwider than a cell (e.g., for a mammalian cell, a channel can have awidth of about 2 µm to about 10 µm). Cellular location on thespatially-barcoded array can be identified using any method describedherein.

In some embodiments, cells can be disposed on an array as describedherein and allowed to migrate. Cell migration in cell migration assayscan be used to measure target phenotypes (e.g., phenotype forinvasiveness). In some embodiments, the cell migration distance can bemeasured and correlated to a biological analyte. Reagents can be addedto the array to facilitate cell migration. For example, the array can becoated with one or more extracellular matrix (ECM) components (e.g.,basement membrane extract (BME), laminin I, collagen I, collagen IV,fibronectin, vitronectin, elastin), a cell culture medium, achemoattractant, a chemorepellant, or a combination thereof. In someembodiments, a reagent such as a chemoattractant or chemorepellant canbe disposed on only a portion of the array, present as a gradient alongthe one or more axis or channels of the array, or a combination thereof.

(VI) Spatially Resolving Analyte Information

In some embodiments, a lookup table (LUT) can be used to associate oneproperty with another property of a feature. These properties include,e.g., locations, barcodes (e.g., nucleic acid barcode molecules),spatial barcodes, optical labels, molecular tags, and other properties.

In some embodiments, a lookup table can associate a nucleic acid barcodemolecule with a feature. In some embodiments, an optical label of afeature can permit associating the feature with a biological particle(e.g., cell or nuclei). The association of a feature with a biologicalparticle can further permit associating a nucleic acid sequence of anucleic acid molecule of the biological particle to one or more physicalproperties of the biological particle (e.g., a type of a cell or alocation of the cell). For example, based on the relationship betweenthe barcode and the optical label, the optical label can be used todetermine the location of a feature, thus associating the location ofthe feature with the barcode sequence of the feature. Subsequentanalysis (e.g., sequencing) can associate the barcode sequence and theanalyte from the sample. Accordingly, based on the relationship betweenthe location and the barcode sequence, the location of the biologicalanalyte can be determined (e.g., in a specific type of cell or in a cellat a specific location of the biological sample).

In some embodiments, a feature can have a plurality of nucleic acidbarcode molecules attached thereto. The plurality of nucleic acidbarcode molecules can include barcode sequences. The plurality ofnucleic acid molecules attached to a given feature can have the samebarcode sequences, or two or more different barcode sequences. Differentbarcode sequences can be used to provide improved spatial locationaccuracy.

As discussed above, analytes obtained from a sample, such as RNA, DNA,peptides, lipids, and proteins, can be further processed. In particular,the contents of individual cells from the sample can be provided withunique spatial barcode sequences such that, upon characterization of theanalytes, the analytes can be attributed as having been derived from thesame cell. More generally, spatial barcodes can be used to attributeanalytes to corresponding spatial locations in the sample. For example,hierarchical spatial positioning of multiple pluralities of spatialbarcodes can be used to identify and characterize analytes over aparticular spatial region of the sample. In some embodiments, thespatial region corresponds to a particular spatial region of interestpreviously identified, e.g., a particular structure of cytoarchitecturepreviously identified. In some embodiments, the spatial regioncorresponds to a small structure or group of cells that cannot be seenwith the naked eye. In some embodiments, a unique molecular identifiercan be used to identify and characterize analytes at a single celllevel.

The analyte can include a nucleic acid molecule, which can be barcodedwith a barcode sequence of a nucleic acid barcode molecule. In someembodiments, the barcoded analyte can be sequenced to obtain a nucleicacid sequence. In some embodiments, the nucleic acid sequence caninclude genetic information associated with the sample. The nucleic acidsequence can include the barcode sequence, or a complement thereof. Thebarcode sequence, or a complement thereof, of the nucleic acid sequencecan be electronically associated with the property (e.g., color and/orintensity) of the analyte using the LUT to identify the associatedfeature in an array.

(VII) Proximity Capture

In some embodiments, two- or three-dimensional spatial profiling of oneor more analytes present in a biological sample can be performed using aproximity capture reaction, which is a reaction that detects twoanalytes that are spatially close to each other and/or interacting witheach other. For example, a proximity capture reaction can be used todetect sequences of DNA that are close in space to each other, e.g., theDNA sequences can be within the same chromosome, but separated by about700 bp or less. As another example, a proximity capture reaction can beused to detect protein associations, e.g., two proteins that interactwith each other. A proximity capture reaction can be performed in situto detect two analytes that are spatially close to each other and/orinteracting with each other inside a cell. Non-limiting examples ofproximity capture reactions include DNA nanoscopy, DNA microscopy, andchromosome conformation capture methods. Chromosome conformation capture(3C) and derivative experimental procedures can be used to estimate thespatial proximity between different genomic elements. Non-limitingexamples of chromatin capture methods include chromosome conformationcapture (3-C), conformation capture-on-chip (4-C), 5-C, ChIA-PET, Hi—C,targeted chromatin capture (T2C). Examples of such methods aredescribed, for example, in Miele et al., Methods Mol Biol. (2009), 464,Simonis et al., Nat. Genet. (2006), 38(11): 1348-54, Raab et al., Embo.J. (2012), 31(2): 330-350, and Eagen et al., Trends Biochem. Sci. (2018)43(6): 469-478, the entire contents of each of which is incorporatedherein by reference.

In some embodiments, the proximity capture reaction includes proximityligation. In some embodiments, proximity ligation can include usingantibodies with attached DNA strands that can participate in ligation,replication, and sequence decoding reactions. For example, a proximityligation reaction can include oligonucleotides attached to pairs ofantibodies that can be joined by ligation if the antibodies have beenbrought in proximity to each oligonucleotide, e.g., by binding the sametarget protein (complex), and the DNA ligation products that form arethen used to template PCR amplification, as described for example inSöderberg et al., Methods. (2008), 45(3): 227-32, the entire contents ofwhich are incorporated herein by reference. In some embodiments,proximity ligation can include chromosome conformation capture methods.

In some embodiments, the proximity capture reaction is performed onanalytes within about 400 nm distance (e.g., about 300 nm, about 200 nm,about 150 nm, about 100 nm, about 50 nm, about 25 nm, about 10 nm, orabout 5 nm) from each other. In general, proximity capture reactions canbe reversible or irreversible.

(VIII) Feature Removal From an Array

A spatially-barcoded array can be contacted with a biological sample tospatially detect analytes present in the biological sample. In someembodiments, the features (e.g., gel pads, beads) can be removed fromthe substrate surface for additional analysis (e.g., imaging,sequencing, or quantification). In some embodiments, the features (e.g.,gel pads, beads) can be removed mechanically (e.g., scraping), by anenzymatic reaction, or by a chemical reaction. In some embodiments, thefeatures (e.g., gel pads, beads) can be aspirated. In some embodiments,after the features are removed (by any method), the features can becombined with a uniquely barcoded bead. In some embodiments, theoligonucleotides within a feature can be ligated or hybridized to thebarcode sequence on the barcoded bead. For example, the spatial barcodeoligonucleotide within a feature can be ligated to the barcode sequenceon the barcoded bead. Additionally, the capture probes can be ligated tothe barcode sequence on the barcoded bead. In some embodiments, thefeatures and the bead can be partitioned. In some embodiments, thefeatures (e.g., gels pads, beads) and the uniquely barcoded bead can bepartitioned into a vesicle. In some embodiments, the vesicle can have alipid bilayer. In some embodiments, the features and the bead can beencapsulated. In some embodiments, the features and the bead can beencapsulated in an oil emulsion. In some embodiments, the features andthe bead can be encapsulated in a water-in-oil emulsion. Oncepartitioned, the features (e.g., gel pads, beads) can be processed forfurther analysis (e.g., sequencing) according to any method describedherein.

(IX) Other Applications

The spatial analysis methods described herein can be used to detect andcharacterize the spatial distribution of one or more haplotypes in abiological sample. As used in the present disclosure, a haplotype isused to describe one or more mutations, DNA variations, polymorphisms ina given segment of the genome, which can be used to classify the geneticsegment, or a collection of alleles or genetic segments containingsingle nucleotide polymorphisms (SNPs). Haplotype association studiesare used to inform a greater understanding of biological conditions. Forexample, identifying and characterizing haplotype variants at orassociated with putative disease loci in humans can provide a foundationfor mapping genetic causes underlying disease susceptibility. The term“locus” (plural “loci”), as used in the art, can be a fixed location ona chromosome, including the location of a gene or a genetic marker,which can contain a plurality of haplotypes, including alleles and SNPs.

Variant haplotype detection is a technique used to identify heterozygouscells in single cell studies. In combination with spatial analysis,variant haplotype detection can further provide novel information on thedistribution of heterozygous cells in biological samples (e.g., tissues)affected by or exhibiting a variety of biological conditions. These datamay reveal causal relationships between variant haplotypes and diseaseoutcomes, to aid in identification of disease-associated variants, or toreveal heterogeneity within a biological sample.

In some embodiments, variant haplotype detection is a technique that canbe used in combination with, in addition to, or as a part of, thespatial analysis methods described herein. Briefly, variant haplotypedetection can include providing inputs for executing an algorithm on acomputer system, and performing an analysis to identify and determinethe spatial distribution of haplotypes. One input can be a plurality ofsequence reads obtained from a two-dimensional spatial array in contactwith a biological sample and subsequently aligned to a genome. Thesequence reads can also contain spatial barcodes with positionalinformation, such that the sequence reads can be mapped to a location onthe biological sample. Other inputs can include electronic data files ofgene sequence variations, or haplotypes, and a reference genome. Foreach locus, the corresponding sequence reads and variant haplotypes arealigned to determine the haplotype identity of each sequence read. Thehaplotype identity and the spatial barcode of the sequence reads arethen categorized to determine the spatial distribution of haplotypeswithin the biological sample. As described above, this spatialdistribution can be used to characterize a biological condition of thesample. In some embodiments, sequence reads are obtained by in situsequencing of the two-dimensional array of positions on the substrate,while in some embodiments, sequence reads are obtained byhigh-throughput sequencing. In some embodiments, other methods forgenerating sequence reads described herein are used, such as paired endsequencings.

In some embodiments, a respective loci in the plurality of loci isbi-allelic and the corresponding set of haplotypes for the respectiveloci consists of a first allele and a second allele. In some suchembodiments, the respective loci includes a heterozygous singlenucleotide polymorphism (SNP), a heterozygous insert, or a heterozygousdeletion.

In some embodiments, analytes captured by any of the spatial analysismethods described herein can be analyzed (e.g., sequenced) via in situsequencing methods. For example, a substrate including a plurality ofcapture probes (e.g., an array), attached either directly or indirectly(e.g., via a feature), that include a spatial barcode and a capturedomain. In some embodiments, the capture domain can be configured tointeract (e.g., hybridize) with an analyte (e.g., mRNA). In someembodiments, a biological sample can be contacted to the array such thatthe capture domain of the capture probe interacts with (e.g.,hybridizes) the analyte. In some embodiments, the capture probe canfunction as a template for a hybridization or ligation reaction with thecaptured analyte. For example, a reverse transcription reaction can beperformed to extend the 3′ end of a capture probe hybridized to theanalyte using any of the exemplary reverse transcriptases describedherein, thereby generating an extended capture probe (e.g., an extendedcapture probe including the spatial barcode and a sequence that iscomplementary to a sequence in the analyte). After the extended captureprobe is synthesized, a second strand that is complementary to theextended capture probe can be synthesized. In some embodiments, secondstrand synthesis can be performed using any of the methods describedherein. In some embodiments, amine-modified nucleotides can be used whengenerating the extended capture probe or the second strand, or both. Forexample, the amine-modified nucleotides can be aminoallyl (aa)-dUTP,aa-dCTP, aa-dGTP, and/or aa-dATP.

In some embodiments, after generation of the extended capture probe, thesecond strand, or both the extended capture probe and/or the secondstrand can be released from the surface of the substrate. For example,the extended capture probe and/or the second strand can be released byany of the methods described herein (e.g., heat or cleavage via acleavage domain). In some embodiments, the amine-modified nucleotidesincorporated into the extended capture probe can be cross- linked to thesurface of a substrate or cross-linked to the biological sample usingits amine-modified nucleotides. In some embodiments, the surface of thesubstrate can be coated in a hydrogel. In some embodiments, the surfaceof the substrate can be coated in a protein matrix. In some embodiments,the cross-linking can be irreversible. In some embodiments, thecross-linked extended capture probe and/or second strand can becircularized. For example, circular template ligation can be performedby a DNA ligase (e.g., T4 DNA ligase) or circular template-free ligationcan be performed by a template independent ligase (e.g., CircLigase). Insome embodiments, the extended capture probe is circularized withCircLigase. In some embodiments, the circularized extended capture probecan be amplified. For example, rolling circle amplification can beperformed with a suitable DNA polymerase (e.g., phi29). In someembodiments, the capture probe includes a functional domain (e.g.,sequencing adapter). In some embodiments, rolling circle amplificationcan be performed with a primer complementary to the functional domain(e.g., sequencing adapter). In some embodiments, the rolling circleamplification can be performed to generate two or more amplicons (e.g.,one or more amplicons including any of the amine-modified nucleotidesdescribed herein). In some embodiments, the two or more ampliconsproduced by the rolling circle amplification can be cross-linked to thesurface of the substrate and/or cross-linked to the biological sample.In some embodiments, the two or more amplicons can be sequenced in situ.The in situ sequencing can be performed by any method described herein(See, Lee, J.H., Fluorescent in situ sequencing (FISSEQ) of RNA for geneexpression profiling, Nat Protoc., 10(3): 442-458,doi:10.1038/nprot.2014.191 (2015), which is incorporated herein byreference). In some embodiments, the two or more amplicons can beimaged.

In some embodiments, spatial analysis by any of the methods describedherein can be performed on ribosomal RNA (rRNA), including, endogenousribosomal RNA (e.g., native to the biological sample), and/or exogenousRNA (e.g., microbial ribosomal RNA and/or viral RNA also present in thebiological sample). As used herein, “metagenomics,” can refer to thestudy of exogenous nucleic acids (e.g., DNA, RNA, or other nucleic acidsdescribed herein) present in a biological sample. As used herein,“spatial metagenomics,” can refer to the study of the spatial locationof exogenous nucleic acid present in a biological sample. Spatialmetagenomics can also refer to the identification of one or more species(e.g., viral or microbial) present in the biological sample and/or thestudy of identifying patterns of proximity (e.g., co-localization)amongst species.

In some embodiments, microbial rRNA can be spatially detected,quantified, and/or amplified from a biological sample. In someembodiments, rRNA (e.g., 16S ribosomal RNA) can be associated with aparticular microbial species. For example, microbial ribosomal RNA(e.g., 16S ribosomal RNA) can be used to identify one or more species ofmicrobe present in the biological sample (See e.g., Kolbert, C.P., andPersing, D.H., Ribosomal DNA sequencing as a tool for identification ofbacterial pathogens, Current Opinion in Microbiology. 2 (3): 299-305.doi:10.1016/S1369-5274(99)80052-6. PMID 10383862 (1999), which isincorporated herein by reference). In some embodiments, identificationof microbial species in proximity to one or more other microbial speciescan be identified.

In some embodiments, a substrate can be covered (e.g., coated) with aphoto-crosslinkable coating (e.g., conditionally dissolvable polymer,e.g., DTT sensitive hydrogel). A biological sample can be contacted withthe photo-crosslinkable coated substrate. In some embodiments, thebiological sample and photo-crosslinkable substrate are assembled into aflow-cell and the photo-crosslinkable polymer can be incubated with thebiological sample. The biological sample can be cross-linked intohydrogel-voxels of defined dimensions using a light source and aphotomask. In some embodiments, the flow-cell can be dismantled andwashed to remove unpolymerized hydrogel. The photo-crosslinkable coatingcan be treated with DTT to yield single-cell partitions or approximatelysingle-cell partitions.

In some embodiments, the single-cell or approximately single-cellpartitions can be encapsulated in a vesicle. The vesicle can contain abarcoded feature (e.g., a bead), and the barcoded feature can contain acapture domain. In some embodiments, the capture domain can capturemicrobial rRNA (e.g., microbial 16S rRNA). In some embodiments, thecaptured microbial rRNA can be amplified and analyzed (e.g., sequenced)by any of the methods described herein. In some embodiments, theamplified and sequenced microbial rRNA can identify microbial speciesand/or patterns of proximity (e.g., co-localization) of one or morespecies.

Alternatively, spatial analysis can be performed on exogenous rRNA(e.g., microbial or viral) with a plurality of capture probes on asubstrate (e.g., an array), wherein the capture probes include a spatialbarcode and a capture domain. In some embodiments, the capture domaincan be configured to interact (e.g., hybridize) with microbial rRNApresent in the biological sample. The capture probe can be configured tointeract with any microbial rRNA. In some embodiments, the capture probeis configured to interact with microbial 16S rRNA. The biological samplecan be treated (e.g., permeabilized) such that the capture domain andthe analyte (e.g., microbial rRNA) interact (e.g., hybridize). In someembodiments, the captured analyte (e.g., microbial rRNA) can be reversetranscribed generating a first strand cDNA, followed by second strandcDNA synthesis as described herein. The first stand cDNA and/or thesecond strand cDNA can include a portion or all of a capture probesequence, or a complement thereof. The capture probe sequence, orcomplement thereof, can include the spatial barcode, or complementthereof. In some embodiments, the first strand cDNA, and optionally, thesecond strand cDNA can be amplified by any method described herein. Theamplified capture probes and analytes can be analyzed (e.g., sequenced)by any method described herein. The spatial information of thespatially-barcoded features can be used to determine the spatiallocation of the captured analytes (e.g., microbial rRNA) in thebiological sample. In some embodiments, the captured analyte canidentify the microbial species present in the biological sample. In someembodiments, the spatial information and identity of microbial speciespresent in the biological sample can be correlated with one another,thus revealing whether certain microbial species may be found inproximity (e.g., co-localize) with one another.

Provided herein are methods for spatially profiling analytes within abiological sample. Profiles of biological samples (e.g., individualcells, populations of cells, tissue sections, etc.) can be compared toprofiles of other cells, e.g., “normal,” or “healthy,” biologicalsamples. In some embodiments of any the methods for spatially profilinganalytes described herein, the method can provide for diagnosis of adisease (e.g., cancer, Alzheimer’s disease, Parkinson’s disease). Insome embodiments of any the methods for spatially profiling analytesdescribed herein, the methods can be used in drug screening. In someembodiments of any the methods for spatially profiling analytesdescribed herein, the methods can be used to perform drug screening withan organoid. In some embodiments of any the methods for spatiallyprofiling analytes described herein, the methods can be used to detectchanges in (e.g., altered) cellular signaling. In some embodiments ofany the methods for spatially profiling analytes described herein, themethods can include the introduction of a pathogen to the biologicalsample and evaluation of the response of the biological sample to thepathogen. In some embodiments of any the methods for spatially profilinganalytes described herein, the methods include exposing the biologicalsample to a perturbation agent (e.g., any of the perturbation agentsdescribed herein) and evaluating the response of the biological sampleto the perturbation agent. In some embodiments of any the methods forspatially profiling analytes described herein, the methods includemonitoring cell differentiation in a biological sample (e.g., anorganoid). In some embodiments of any the methods for spatiallyprofiling analytes described herein, the methods include analyzingtissue morphogenesis. In some embodiments of any the methods forspatially profiling analytes described herein, the methods includeidentifying spatial heterogeneity in a biological sample (e.g.,identifying different cell types or populations in a biological sample).In some embodiments of any the methods for spatially profiling analytesdescribed herein, the methods include analyzing the spatiotemporal order(e.g., timing) of molecular events. For example, the methods forspatially profiling analytes can include monitoring expression levelsover the course of a disease.

The methods provided herein can also be used to determine a relativelevel of inflammation in a subject (e.g., determine an inflammatoryscore) or a subject’s response to treatment or the development ofresistance to treatment. The methods described herein can also be usedto identify candidate targets for potential therapeutic interventionand/or to identify biomarkers associated with different disease statesin a subject.

(H) Quality Control (I) Control Sample

As used herein, the term “control sample” typically refers to asubstrate that is insoluble in aqueous liquid and that allows for anaccurate and traceable positioning of test analytes on the substrate.The control sample can be any suitable substrate known to the personskilled in the art. Exemplary control samples comprise a semi-porousmaterial. Non-limiting examples of a semi-porous material include anitrocellulose membrane, a hydrogel, and a nylon filter.

A control sample can be of any appropriate dimension or volume (e.g.,size or shape). In some embodiments, a control sample is a regular shape(e.g., a square, circle, or a rectangle). In some embodiments, a surfaceof a control sample has any appropriate form or format. For example, thesurface of a control sample can be flat or curved (e.g., convexly orconcavely curved towards the area where the interaction between thesubstrate and the control sample takes place). In some embodiments, acontrol sample has rounded corners (e.g., for increased safety orrobustness). In some embodiments, a control sample has one or morecut-off corners (e.g., for use with a slide clamp or cross-table).

A control sample can comprise a plurality of test analytes. In someembodiments, the members of the plurality of test analytes are disposedon the substrate in a known amount and in a known location. For example,a plurality of test analytes are disposed at a known amount on thecontrol sample at one or more locations. In some embodiments, theplurality of test analytes are disposed on the substrate in a definedpattern (e.g., an x-y grid pattern). In some embodiments, the definedpattern includes one or more locations or spots.

In some embodiments, each location comprises a plurality of the samespecies of test analyte. In some embodiments, each location comprises aplurality of one or more different species of test analytes. In someembodiments, each location on the control sample represents a differentregion of a biological sample, e.g., a tissue sample. In someembodiments, an area on the control sample that does not comprise aplurality of test analytes represents an area where no biological sampleis present.

In some embodiments, the plurality of test analytes comprises one ormore test analytes, e.g., a first test analyte, a second test analyte, athird test analyte, a fourth test analyte, etc. In some embodiments, theplurality of test analytes comprises nucleic acids. In some embodiments,each location or feature comprises a population of nucleic acidsequences. In some embodiments, the nucleic acid sequence of a firsttest analyte differs from the nucleic acid sequence of a second testanalyte by a single nucleic acid residue. In some embodiments, eachlocation or feature comprises a population of RNA transcripts and one ormore specific surface marker proteins or one or more CRISPR guide RNAs.In some embodiments, the plurality of test analytes comprises abacterial artificial chromosomes (BAC). In some embodiments, eachlocation on the control sample comprises a unique blend of BACs. In someembodiments, proteins are cross-linked to the BACs, for example, tomimic histone binding on DNA.

In some embodiments, the concentration of a first test analyte differsfrom the concentration of a second test analyte at a different locationor feature on the control sample. In some embodiments, the first testanalyte and the second test analyte comprise an identical nucleotidesequence.

(II) Spatial RNA Integrity Number (sRIN)

As used herein, the term “spatial RNA Integrity Number” or “sRIN” refersto the in situ indication of RNA quality based on an integrity score.Higher sRIN scores correlate with higher data quality in the spatialprofiling assays described herein. For example, a first biologicalsample with a high sRIN score will have higher data quality compared toa second biological sample with sRIN score lower than the firstbiological sample. In some embodiments, a sRIN is calculated for atissue section, one or more regions of a tissue section, or a singlecell.

In some embodiments, one or more sRINs for a given biological sample(e.g., tissue section, one or more regions of a tissue, or a singlecell) are calculated by: (a) providing (i) a spatial array including aplurality of capture probes on a substrate, where a capture probecomprises a capture domain and (ii) a tissue stained with a histologystain (e.g., any of the stains described herein); (b) contacting thespatial array with the biological sample (e.g., tissue); (c) capturing abiological analyte (e.g., an 18S rRNA molecule) from the biologicalsample (e.g., tissue) with the capture domain; (d) generating a cDNAmolecule from the captured biological analyte (e.g., 18S rRNA); (e)hybridizing one or more labeled oligonucleotide probes to the cDNA; (f)imaging the labeled cDNA and the histology stain (e.g., any of thestains described herein), and (g) generating a spatial RNA integritynumber for a location in the spatial array, wherein the spatial RNAintegrity number comprises an analysis of a labeled cDNA image and ahistology stain (e.g., any of the stains described herein) image for thelocation.

In some embodiments, the biological sample (e.g., tissue) is stainedwith a histology stain. As used herein, a “histology stain” can be anystain described herein. For example, the biological sample can bestained with IF/IHC stains described herein. For example, the biologicalsample (e.g., tissue) can be stained with Hematoxylin & Eosin (“H&E”).In some embodiments, the biological sample (e.g., tissue) is stainedwith a histology stain (e.g., any of the stains described herein)before, contemporaneously with, of after labelling of the cDNA withlabeled oligonucleotide probes. In some embodiments, the stainedbiological sample can be, optionally, destained (e.g., washed in HCl).For example, Hematoxylin, from the H&E stain, can be optionally removedfrom the biological sample by washing in dilute HCl (0.01 M) prior tofurther processing. In some embodiments, the stained biological samplecan be optionally destained after imaging and prior to permeabilization.

In some embodiments, the spatial array includes a plurality of captureprobes immobilized on a substrate where the capture probes include atleast a capture domain. In some embodiments, the capture domain includesa poly(T) sequence. For example, a capture domain includes a poly(T)sequence that is capable of capturing an 18S rRNA transcript from abiological sample.

In some embodiments, calculating one or more spatial RNA IntegrityNumbers for a biological sample includes hybridizing at least one (e.g.,at least two, at least three, at least four, or at least five) labeledoligonucleotide probes to the cDNA generated from the 18 s rRNA. In someembodiments, a labeled oligonucleotide probe includes a sequence that iscomplementary to a portion of the 18S cDNA. In some embodiments, fourlabeled oligonucleotide probes (P1—P4) are designed to hybridize at fourdifferent locations spanning the entire gene body of the 18S rRNA. Insome embodiments, a labeled oligonucleotide probe can include any of thedetectable labels as described herein. For example, an oligonucleotidelabeled probe can include a fluorescent label (e.g., Cy3). In someembodiments, one or more of the labeled oligonucleotide probes designedwith complementarity to different locations within the 18S cDNA sequenceinclude the same detectable label. For example, four labeledoligonucleotide probes, (P1—P4) each designed to have complementarity toa different location within the 18S cDNA sequence can all have the samedetectable label (e.g., Cy3). In some embodiments, one or more of thelabeled oligonucleotide probes designed with complementarity todifferent locations within the 18S cDNA sequence include a differentdetectable label. For example, four labeled oligonucleotide probes,(P1—P4) each designed to have complementarity to a different locationwithin the 18S cDNA sequence can include different detectable labels.

In some embodiments, determining a spatial RNA Integrity Number for abiological sample (e.g., tissue section, one or more regions of atissue, or a single cell) includes analyzing the images taken from aspatial array and a histology stain (e.g., any of the stains describedherein) for the same location. For example, for the spatial array, allimages are generated by scanning with a laser (e.g., a 532 nmwavelength) after the fluorescently labeled (e.g., Cy3) oligonucleotideprobes have been hybridized to the 18S cDNA. One image is generated perprobe (P1—P4) and one image is generated where no fluorescently labeledprobes were hybridized (P0). Normalization of Fluorescence Units (FU)data is performed by subtraction of the auto-fluorescence recorded withP0 and division with P1. After alignment, the five images (one imagefrom each probe, P1—P4, and one image from an area without bound probe)are loaded into a script. The script generates two different plots, oneheat-map of spatial RIN values and one image alignment error plot, whichcombines the histology stain (e.g., any of the stains described herein)image. The image alignment error plot is used to visualize which pixelsand positions should be excluded from the analysis due to alignmenterrors between the images from P0—P4.

III. General Spatial Cell-Based Analytical Methodology (a) Barcoding aBiological Sample

In some embodiments, provided herein are methods and materials forattaching and/or introducing a molecule (e.g., a peptide, a lipid, or anucleic acid molecule) having a barcode (e.g., a spatial barcode) to abiological sample (e.g., to a cell in a biological sample) for use inspatial analysis. In some embodiments, a plurality of molecules (e.g., aplurality of nucleic acid molecules) having a plurality of barcodes(e.g., a plurality of spatial barcodes) are introduced to a biologicalsample (e.g., to a plurality of cells in a biological sample) for use inspatial analysis.

FIG. 18 is a schematic diagram depicting cell tagging using eithercovalent conjugation of an analyte binding moiety to a cell surface ornon-covalent interactions with cell membrane elements. FIG. 18 listsnon-exhaustive examples of a covalent analyte binding moiety/cellsurface interactions, including protein targeting, amine conjugationusing NHS chemistry, cyanuric chloride, thiol conjugation via maleimideaddition, as well as targeting glycoproteins/glycolipids expressed onthe cell surface via click chemistry. Non-exhaustive examples ofnon-covalent interactions with cell membrane elements include lipidmodified oligos, biocompatible anchor for cell membrane (BAM, e.g.,oleyl-PEG-NHS), lipid modified positive neutral polymer, and antibody tomembrane proteins. A cell tag can be used in combination with an analytecapture agent and cleavable or non-cleavable spatially-barcoded captureprobes for spatial and multiplexing applications.

In some embodiments, a plurality of molecules (e.g., a plurality oflipid or nucleic acid molecules) having a plurality of barcodes (e.g., aplurality of spatial barcodes) are introduced to a biological sample(e.g., to a plurality of cells in a biological sample) for use inspatial analysis, wherein the plurality of molecules are introduced tothe biological sample in an arrayed format. In some embodiments, aplurality of molecules (e.g., a plurality of lipid or nucleic acidmolecules) having a plurality of barcodes are provided on a substrate(e.g., any of the variety of substrates described herein) in any of thevariety of arrayed formats described herein, and the biological sampleis contacted with the molecules on the substrate such that the moleculesare introduced to the biological sample. In some embodiments, themolecules that are introduced to the biological sample are cleavablyattached to the substrate, and are cleaved from the substrate andreleased to the biological sample when contacted with the biologicalsample. In some embodiments, the molecules introduced to the biologicalsample are covalently attached to the substrate prior to cleavage. Insome embodiments, the molecules that are introduced to the biologicalsample are non-covalently attached to the substrate (e.g., viahybridization), and are released from the substrate to the biologicalsample when contacted with the biological sample.

In some embodiments, a plurality of molecules (e.g., a plurality oflipid or nucleic acid molecules) having a plurality of barcodes (e.g., aplurality of spatial barcodes) are migrated or transferred from asubstrate to cells of a biological sample. In some embodiments,migrating a plurality of molecules from a substrate to cells of abiological sample includes applying a force (e.g., mechanical,centrifugal, or electrophoretic) to the substrate and/or the biologicalsample to facilitate migration of the plurality of molecules from thesubstrate to the biological sample.

In some embodiments of any of the spatial analysis methods describedherein, physical force is used to facilitate attachment to orintroduction of a molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) into a biological sample (e.g., a cellpresent in a biological sample). As used herein, “physical force” refersto the use of a physical force to counteract the cell membrane barrierin facilitating intracellular delivery of molecules. Examples ofphysical force instruments and methods that can be used in accordancewith materials and methods described herein include the use of a needle,ballistic DNA, electroporation, sonoporation, photoporation,magnetofection, hydroporation, and combinations thereof.

(I) Introducing a Cell-Tagging Agent to the Surface of a Cell

In some embodiments, biological samples (e.g., cells in a biologicalsample) can be labelled using cell-tagging agents where the cell-taggingagents facilitate the introduction of the molecules (e.g., nucleic acidmolecules) having barcodes (e.g., spatial barcodes) into the biologicalsample (e.g., into cells in a biological sample). As used herein, theterm “cell-tagging agent” refers to a molecule having a moiety that iscapable of attaching to the surface of a cell (e.g., thus attaching thebarcode to the surface of the cell) and/or penetrating and passingthrough the cell membrane (e.g., thus introducing the barcode to theinterior of the cell). In some embodiments, a cell-tagging agentincludes a barcode (e.g., a spatial barcode). The barcode of a barcodedcell-tagging agent can be any of the variety of barcodes describedherein. In some embodiments, the barcode of a barcoded cell-taggingagent is a spatial barcode. In some embodiments, a cell-tagging agentcomprises a nucleic acid molecule that includes the barcode (e.g., thespatial barcode). In some embodiments, the barcode of a barcodedcell-tagging agent identifies the associated molecule, where eachbarcode is associated with a particular molecule. In some embodiments,one or more molecules are applied to a sample. In some embodiments, anucleic acid molecule that includes the barcode is covalently attachedto the cell-tagging agent. In some embodiments, a nucleic acid moleculethat includes the barcode is non-covalently attached to the cell-taggingagent. A non-limiting example of non-covalent attachment includeshybridizing the nucleic acid molecule that includes the barcode to anucleic acid molecule on the cell-tagging agent (which nucleic acidmolecule on the cell-tagging agent can be bound to the cell-taggingagent covalently or non-covalently). In some embodiments, a nucleic acidmolecule attached to a cell-tagging agent that includes a barcode (e.g.,a spatial barcode) also includes one or more additional domains. Suchadditional domains include, without limitation, a PCR handle, asequencing priming site, a domain for hybridizing to another nucleicacid molecule, and combinations thereof.

In some embodiments, a cell-tagging agent attaches to the surface of acell. When the cell-tagging agent includes a barcode (e.g., a nucleicacid that includes a spatial barcode), the barcode is also attached tothe surface of the cell. In some embodiments of any of the spatialanalysis methods described herein, a cell-tagging agent attachescovalently to the cell surface to facilitate introduction of the spatialanalysis reagents. In some embodiments of any of the spatial analysismethods described herein, a cell-tagging agent attaches non-covalentlyto the cell surface to facilitate introduction of the spatial analysisreagents.

In some embodiments, once a cell or cells in a biological sample isspatially tagged with a cell-tagging agent(s), spatial analysis ofanalytes present in the biological sample is performed. In someembodiments, such spatial analysis includes dissociating thespatially-tagged cells of the biological sample (or a subset of thespatially-tagged cells of the biological sample) and analyzing analytespresent in those cells on a cell-by-cell basis. Any of a variety ofmethods for analyzing analytes present in cells on a cell-by-cell basiscan be used. Non-limiting examples include any of the variety of methodsdescribed herein and methods described in PCT Application PublicationNo. WO 2019/113533A1, the content of which is incorporated herein byreference in its entirety. For example, the spatially-tagged cells canbe encapsulated with beads comprising one or more nucleic acid moleculeshaving a barcode (e.g., a cellular barcode) (e.g., an emulsion). Thenucleic acid present on the bead can have a domain that hybridizes to adomain on a nucleic acid present on the tagged cell (e.g., a domain on anucleic acid that is attached to a cell-tagging agent), thus linking thespatial barcode of the cell to the cellular barcode of the bead. Oncethe spatial barcode of the cell and the cellular barcode of the bead arelinked, analytes present in the cell can be analyzed using captureprobes (e.g., capture probes present on the bead). This allows thenucleic acids produced (using these methods) from specific cells to beamplified and sequenced separately (e.g., within separate partitions ordroplets).

In some embodiments, once a cell or cells in a biological sample isspatially tagged with a cell-tagging agent(s), spatial analysis ofanalytes present in the biological sample is performed in which thecells of the biological sample are not dissociated into single cells. Insuch embodiments, various methods of spatial analysis such as any ofthose provided herein can be employed. For example, once a cell or cellsin a biological sample is spatially tagged with a cell-tagging agent(s),analytes in the cells can be captured and assayed. In some embodiments,cell-tagging agents include both a spatial barcode and a capture domainthat can be used to capture analytes present in a cell. For example,cell-tagging agents that include both a spatial barcode and a capturedomain can be introduced to cells of the biological sample in a way suchthat locations of the cell-tagging agents are known (or can bedetermined after introducing them to the cells). One non-limitingexample of introducing cell-tagging agents to a biological sample is toprovide the cell-tagging agents in an arrayed format (e.g., arrayed on asubstrate such as any of the variety of substrates and arrays providedherein), where the positions of the cell-tagging agents on the array areknown at the time of introduction (or can be determined afterintroduction). The cells can be permeabilized as necessary (e.g., usingpermeabilization agents and methods described herein), reagents foranalyte analysis can be provided to the cells (e.g., a reversetranscriptase, a polymerase, nucleotides, etc., in the case where theanalyte is a nucleic acid that binds to the capture probe), and theanalytes can be assayed. In some embodiments, the assayed analytes(and/or copies thereof) can be released from the substrate and analyzed.In some embodiments, the assayed analytes (and/or copies thereof) areassayed in situ.

Non-limiting examples of cell-tagging agents and systems that attach tothe surface of a cell (e.g., thus introducing the cell-tagging agent andany barcode attached thereto to the exterior of the cell) that can beused in accordance with materials and methods provided herein forspatially analyzing an analyte or analytes in a biological sampleinclude: lipid tagged primers / lipophilic-tagged moieties, positive orneutral oligo-conjugated polymers, antibody-tagged primers,streptavidin-conjugated oligonucleotides, dye-tagged oligonucleotides,click-chemistry, receptor-ligand systems, covalent binding systems viaamine or thiol functionalities, and combinations thereof.

(ii) Introducing a Cell-Tagging Agent to the Interior of a CellNon-limiting examples of cell-tagging agents and systems that penetrateand/or pass through the cell membrane (e.g., thus introducing thecell-tagging agent and any barcode attached thereto to the interior ofthe cell) that can be used in accordance with materials and methodsprovided herein for spatially profiling an analyte or analytes in abiological sample include: a cell-penetrating agent (e.g., acell-penetrating peptide), a nanoparticle, a liposome, a polymersome, apeptide-based chemical vector, electroporation, sonoporation, lentiviralvectors, retroviral vectors, and combinations thereof.

FIG. 19 is a schematic showing an exemplary cell tagging method.Non-exhaustive examples of oligo delivery vehicles may include a cellpenetrating peptide or a nanoparticle. Non-exhaustive examples of thedelivery systems can include lipid-based polymeric and metallicnanoparticles or oligos that can be conjugated or encapsulated withinthe delivery system. The cell tag can be used in combination with acapture agent barcode domain and a cleavable or non-cleavablespatially-barcoded capture probes for spatial and multiplexingapplications.

1. Cell-Penetrating Agent

In some embodiments of any of the spatial profiling methods describedherein, identification of a biological analyte by a molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) and acapture domain is facilitated by a cell-penetrating agent. In someembodiments, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) and a capture domain is coupled to acell-penetrating agent, and the cell-penetrating agent allows themolecule to interact with an analyte inside the cell. A“cell-penetrating agent” as used herein can refer to an agent capable offacilitating the introduction of a molecule (e.g., a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) and a capturedomain into a cell of a biological sample (see, e.g., Lovatt et al. NatMethods. 2014 Feb;11(2):190-6, which is incorporated herein by referencein its entirety). In some embodiments, a cell-penetrating agent is acell-penetrating peptide. A “cell-penetrating peptide” as used hereinrefers to a peptide (e.g., a short peptide, e.g., a peptide not usuallyexceeding 30 residues) that has the capacity to cross cellularmembranes. In some embodiments, cell-penetrating agents or cellpenetrating peptides may be covalently or non-covalently coupled to amolecule (e.g., a barcoded nucleic acid molecule), likely at the 5′ endof the molecule. A cell-penetrating peptide may direct the barcodednucleic acid molecule to a specific organelle.

In some embodiments of any of the spatial profiling methods describedherein, a cell-penetrating peptide coupled to a molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) and acapture domain can cross a cellular membrane using an energy dependentor an energy independent mechanism. For example, a cell-penetratingpeptide can cross a cellular membrane through direct translocationthrough physical perturbation of the plasma membrane, endocytosis (e.g.,mediated via clathrin), adaptive translocation, pore-formation,electroporation-like permeabilization, and/or entry at microdomainboundaries. Non-limiting examples of a cell-penetrating peptide include:penetratin, tat peptide, pVEC, transportan, MPG, Pep-1, a polyargininepeptide, MAP, R6W3, (D-Arg)9, Cys(Npys)—(D—Arg)9, Anti-BetaGamma (MPS -Phosducin - like protein C terminus), Cys(Npys) antennapedia,Cys(Npys)-(Arg)9, Cys(Npys)-TAT (47-57), HIV-1 Tat (48-60), KALA,mastoparan, penetratin-Arg, pep-1-cysteamine, TAT(47-57)GGG-Cys(Npys),Tat-NR2Bct, transdermal peptide, SynB1, SynB3, PTD-4, PTD-5, FHVCoat-(35-49), BMV Gag-(7-25), HTLV-II Rex-(4-16), R9-tat, SBP, FBP, MPG,MPG(ΔNLS), Pep-2, MTS, plsl, and a polylysine peptide (see, e.g.,Bechara et al. FEBS Lett. 2013 Jun 19;587(12):1693-702, which isincorporated by reference herein in its entirety).

In some embodiments, there could be two orientations forcell-penetrating peptide (CPP) conjugation. For example, one orientationcan be (N-terminus)-CPP-Cys-(C-terminus)-linker-NH2C6-5′-oligo-3′;3′-oligo-5′—NH2C6—linker-(N-terminus)-Cys-CPP-(C-terminus). The methodsherein can be performed with other CPP conjugations and orientations.

2. Nanoparticles

In some embodiments of any of the spatial profiling methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by an inorganic particle (e.g., a nanoparticle).In some embodiments, a molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) and a capture domain is coupled to aninorganic particle (e.g., a nanoparticle), and the molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) and acapture domain uses the nanoparticle to get access to analytes insidethe cell. Non-limiting examples of nanoparticles that can be used inembodiments herein to deliver a molecule (e.g., a nucleic acid molecule)having a barcode (e.g., a spatial barcode) and a capture domain into acell and/or cell bead include inorganic nanoparticles prepared frommetals, (e.g., iron, gold, and silver), inorganic salts, and ceramics(e.g., phosphate or carbonate salts of calcium, magnesium, or silicon).The surface of a nanoparticle can be coated to facilitate binding of themolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) and a capture domain, or the surface can be chemicallymodified to facilitate attachment of the molecule (e.g., a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) and a capturedomain. Magnetic nanoparticles (e.g., supermagnetic iron oxide),fullerenes (e.g., soluble carbon molecules), carbon nanotubes (e.g.,cylindrical fullerenes), quantum dots, and supramolecular systems canalso be used.

3. Liposomes

In some embodiments of any of the spatial analysis methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by a liposome. Various types of lipids, includingcationic lipids, can be used in liposome delivery. In some cases, amolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) and a capture domain is delivered to a cell via a lipidnano-emulsion. A lipid emulsion refers to a dispersion of one immiscibleliquid in another stabilized by emulsifying agent. Labeling cells cancomprise use of a solid lipid nanoparticle.

4. Polymersomes

In some embodiments of any of the spatial analysis methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by a polymersome. In some embodiments, a molecule(e.g., a nucleic acid molecule) having a barcode (e.g., a spatialbarcode) and a capture domain is contained in the polymersome, and themolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) and a capture domain uses the polymersome to get accessto analytes inside the cell. A “polymersome” as referred to herein is anartificial vesicle. For example, a polymersome can be a vesicle similarto a liposome, but the membrane comprises amphiphilic synthetic blockcopolymers (see, e.g., Rideau et al. Chem. Soc. Rev., 2018, 47,8572-8610, which is incorporated by reference herein in its entirety).In some embodiments, polymersomes comprise di-(AB) or tri-blockcopolymers (e.g., ABA or ABC), where A and C are a hydrophilic block andB is a hydrophobic block. In some embodiments, a polymersome comprisespoly(butadiene)-b-poly(ethylene oxide), poly(ethylethylene)-b-poly(ethylene oxide), polystyrene-b-poly(ethylene oxide),poly(2-vinylpyridine)-b- poly(ethylene oxide),polydimethylsiloxane-b-poly(ethylene oxide), polydimethylsiloxane-g-poly(ethylene oxide), polycaprolactone-b-poly(ethylene oxide),polyisobutylene-b-poly(ethylene oxide), polystyrene-b- polyacrylic acid,polydimethylsiloxane-b- poly-2-methyl-2-oxazoline, or a combinationthereof (wherein b=block and g=grafted).

5. Peptide-Based Chemical Vectors

In some embodiments of any of the spatial analysis methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by a peptide-based chemical vector, e.g., acationic peptide-based chemical vector. Cationic peptides can be rich inbasic residues like lysine and/or arginine. In some embodiments of anyof the spatial analysis methods described herein, capture of abiological analyte by a molecule (e.g., a nucleic acid molecule) havinga barcode (e.g., a spatial barcode) and a capture domain is facilitatedby a polymer-based chemical vector. Cationic polymers, when mixed with amolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) and a capture domain, can form nanosized complexescalled polyplexes. Polymer based vectors can comprise natural proteins,peptides and/or polysaccharides. Polymer based vectors can comprisesynthetic polymers. In some embodiments, a polymer-based vectorcomprises polyethylenimine (PEI). PEI can condense DNA intopositively-charged particles, which bind to anionic cell surfaceresidues and are brought into the cell via endocytosis. In someembodiments, a polymer-based chemical vector comprises poly(L)-lysine(PLL), poly (DL-lactic acid) (PLA), poly (DL-lactide-co-glycoside)(PLGA), polyornithine, polyarginine, histones, protamines, or acombination thereof. Polymer-based vectors can comprise a mixture ofpolymers, for example, PEG and PLL. Other non-limiting examples ofpolymers include dendrimers, chitosans, synthetic amino derivatives ofdextran, and cationic acrylic polymers.

6. Electroporation

In some embodiments of any of the spatial analysis methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by electroporation. With electroporation, abiological analyte by a molecule (e.g., a nucleic acid molecule) havinga barcode (e.g., a spatial barcode) and a capture domain can enter acell through one or more pores in the cellular membrane formed byapplied electricity. The pore of the membrane can be reversible based onthe applied field strength and pulse duration.

7. Sonoporation

In some embodiments of any of the spatial analysis methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by sonoporation. Cell membranes can be temporarilypermeabilized using sound waves, allowing cellular uptake of abiological analyte by a molecule (e.g., a nucleic acid molecule) havinga barcode (e.g., a spatial barcode) and a capture domain.

8. Lentiviral Vectors and Retroviral Vectors

In some embodiments of any of the spatial analysis methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by vectors. For example, a vector as describedherein can be an expression vector where the expression vector includesa promoter sequence operably linked to the sequence encoding themolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) and a capture domain. Non-limiting examples of vectorsinclude plasmids, transposons, cosmids, and viral vectors (e.g., anyadenoviral vectors (e.g., pSV or pCMV vectors), adeno-associated virus(AAV) vectors, lentivirus vectors, and retroviral vectors), and anyGateway® vectors. A vector can, for example, include sufficientcis-acting elements for expression where other elements for expressioncan be supplied by the host mammalian cell or in an in vitro expressionsystem. Skilled practitioners will be capable of selecting suitablevectors and mammalian cells for introducing any of spatial profilingreagents described herein.

9. Other Methods and Cell-Tagging Agents for Intracellular Introductionof a Molecule

In some embodiments of any of the spatial analysis methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by the use of a needle, for example for injection(e.g., microinjection), particle bombardment, photoporation,magnetofection, and/or hydroporation. For example, with particlebombardment, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) and a capture domain can be coated with heavymetal particles and delivered to a cell at a high speed. Inphotoporation, a transient pore in a cell membrane can be generatedusing a laser pulse, allowing cellular uptake of a molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) and acapture domain. In magnetofection, a molecule (e.g., a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) and a capturedomain can be coupled to a magnetic particle (e.g., magneticnanoparticle, nanowires, etc.) and localized to a target cell via anapplied magnetic field. In hydroporation, a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain can be delivered to a cell and/or cell bead via hydrodynamicpressure.

(III) Lipid Tagged Primers / Lipophilic-Tagged Moieties

In some embodiments of any of the spatial profiling methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) is coupled to a lipophilic molecule. In someembodiments, the lipophilic molecule enables delivery of the lipophilicmolecule to the cell membrane or the nuclear membrane. In someembodiments, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) coupled to a lipophilic molecule can associatewith and/or insert into lipid membranes such as cell membranes andnuclear membranes. In some cases, the insertion can be reversible. Insome cases, the association between the lipophilic molecule and the cellmay be such that the cell retains the lipophilic molecule (e.g., andassociated components, such as nucleic acid barcode molecules) duringsubsequent processing (e.g., partitioning, cell permeabilization,amplification, pooling, etc.). In some embodiments, a molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode)coupled to a lipophilic molecule may enter into the intracellular spaceand/or a cell nucleus.

Non-limiting examples of lipophilic molecules that can be used inembodiments described herein include sterol lipids such as cholesterol,tocopherol, steryl, palmitate, lignoceric acid, and derivatives thereof.In some embodiments, the lipophilic molecules are neutral lipids thatare conjugated to hydrophobic moieties (e.g., cholesterol, squalene, orfatty acids) (See Raouane et al. Bioconjugate Chem., 23(6):1091-1104(2012) which is herein incorporated by reference in its entirety). Insome embodiments, a molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) may be attached to the lipophilicmoiety via a linker, using covalent or direct attachment. In someembodiments, the linker is a tetra-ethylene glycol (TEG) linker. Otherexemplary linkers include, but are not limited to, Amino Linker C6,Amino Linker C12, Spacer C3, Spacer C6, Spacer C12, Spacer 9, and Spacer18. In some embodiments, a molecule (e.g., a nucleic acid molecule)having a barcode (e.g., a spatial barcode) is indirectly coupled (e.g.,via hybridization or ligand-ligand interactions, such asbiotin-streptavidin) to a lipophilic molecule. In some embodiments, thelipophilic moiety can be attached to a capture probe, spatial barcode,or other DNA sequence, at either the 5′ or 3′ end of the specified DNAsequence. In some embodiments, the lipophilic moiety can be coupled to acapture probe, spatial barcode, or other DNA sequence in alipid-dependent manner. Other lipophilic molecules that may be used inaccordance with methods provided herein include amphiphilic moleculeswherein the headgroup (e.g., charge, aliphatic content, and/or aromaticcontent) and/or fatty acid chain length (e.g., C12, C14, C16, or C18)can be varied. For instance, fatty acid side chains (e.g., C12, C14,C16, or C18) can be coupled to glycerol or glycerol derivatives (e.g.,3-t-butyldiphenylsilylglycerol), which can also comprise, e.g., acationic head group. In some embodiments, a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) disclosedherein can then be coupled (either directly or indirectly) to theseamphiphilic molecules. In some embodiments, a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) coupled to anamphiphilic molecule may associate with and/or insert into a membrane(e.g., a cell, cell bead, or nuclear membrane). In some cases, anamphiphilic or lipophilic moiety may cross a cell membrane and provide amolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) to an internal region of a cell and/or cell bead.

In some embodiments, additives can be added to supplement lipid-basedmodifications. In some embodiments, the additive is low densitylipoprotein (LDL). In some embodiments, the additive is the cholesteroltrafficking inhibitor U-18666A. In some embodiments, U-18666A inhibitscholesterol transport from late endosomes at micromolar concentrationsand/or lysosomes to the endoplasmic reticulum (ER) at nanomolarconcentrations. In some embodiments, U-18666A can inhibit oxidosqualenecyclase, a key enzyme in the cholesterol biosynthesis pathway, atsufficiently high concentrations (e.g., at or about >0.5 mM).

In some embodiments, wherein the molecule (e.g., with a nucleic acidsequence) has an amino group within the molecule, the molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) and anamino group can be coupled to an amine-reactive lipophilic molecule. Forexample, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) and an amino group can be conjugated toDSPE-PEG(2000)-cyanuric chloride(1,2-distearoyl-sn-glycero-3-phosphoethanolamine—N—[cyanur(polyethyleneglycol)-2000]).

In some embodiments, a cell-tagging agent can attach to a surface of acell through a combination of lipophilic and covalent attachment. Forexample, a cell-tagging agent can include an oligonucleotide attached toa lipid to target the oligonucleotide to a cell membrane, and an aminegroup that can be covalently linked to a cell surface protein(s) via anynumber of chemistries described herein. In these embodiments, the lipidcan increase the surface concentration of the oligonucleotide and canpromote the covalent reaction.

As used herein, an “anchor oligonucleotide” and/or “co-anchoroligonucleotide” can include a lipid-conjugated oligonucleotide, whereinthe lipid is capable of becoming embedded within a cell membrane. Insome embodiments, the lipid capable of becoming embedded within a cellmembrane includes but is not limited to, sterol lipids such ascholesterol, tocopherol, steryl, palmitate, lignoceric acid, andderivatives thereof. In some embodiments, the sterol lipid of the anchoroligonucleotide and/or co-anchor oligonucleotide can be attached toeither the 5′ or 3′ end of the oligonucleotide portion. In someembodiments, the anchor oligonucleotide and/or the co-anchoroligonucleotide can integrate into the cell membrane of a cell in abiological sample (e.g., the sterol lipid of the anchor oligonucleotideand/or co-anchor oligonucleotide).

In some embodiments, a sterol lipid (e.g., lignoceric acid) anchoroligonucleotide is attached to the 5′ end of the oligonucleotide. Insome embodiments, the anchor oligonucleotide can have a constantsequence. In some embodiments the constant sequence of the anchoroligonucleotide can be about 15 to about 30 nucleotides long. In someembodiments, the anchor oligonucleotide can have an additional domain 3′to the constant sequence. In some embodiments, the additional domain canbe an adapter sequence (e.g., sequencing adapter). In some embodiments,the adapter sequence can be about 15 to about 35 nucleotides long.

In some embodiments, the lipid (e.g., sterol lipid) of the co-anchoroligonucleotide (e.g., palmitic acid), is attached to the 3′ end of theoligonucleotide. In some embodiments, the co-anchor oligonucleotide canhave a constant sequence. For example, the constant sequence of theco-anchor oligonucleotide can be a reverse complement of the constantsequence from the anchor oligonucleotide. In some embodiments, theconstant sequence of the anchor oligonucleotide and the constantsequence of the co-anchor oligonucleotide can bind (e.g., hybridize) toeach other. In some embodiments, the lipid (e.g., sterol lipid) of theanchor oligonucleotide and the co-anchor oligonucleotide can integrateinto a cell membrane in the biological sample and the respectiveconstant sequences can hybridize to each other at the same time. In someembodiments, a barcoded oligonucleotide, which can include severaldomains, can be introduced to the integrated anchor oligonucleotide andco-anchor oligonucleotide hybridized to each other. The barcodedoligonucleotide can include, in a 5′ to 3′ direction, a functionaldomain (e.g., a sequencing adapter domain), a unique molecularidentifier, a sample barcode, a second unique molecular identifier, andthe reverse complement of a constant sequence. For example, aftertagging a cell with any of the cell-tagging agents described herein thecells can be partitioned (e.g., encapsulated in a vesicle) with abarcoded feature (e.g., a bead). In some embodiments, the reversecomplement of the constant sequence of the barcoded oligonucleotide caninteract (e.g., hybridize) with the constant sequence (e.g., a portionof the sequence) on the barcoded feature.

(IV) Intracellular Cleavage Domain

As used herein, capture probes can optionally include an “intracellularcleavage domain,” wherein one or more segments or regions of the captureprobe (e.g., capture domains, spatial barcodes, and/or UMIs) can bereleasably or cleavably attached to one or more other segments orregions of the capture probe, such as a cell-penetrating agent, suchthat the capture domain, spatial barcode, and/or UMI can be released orbe releasable through cleavage of a linkage between the capture domain,spatial barcode, and/or UMI and the cell-penetrating agent and/or cellpenetration tag. In some embodiments, the cleavage of the linkagebetween the capture domain, spatial barcode, and/or UMI and thecell-penetrating agent is induced in an intracellular environment (e.g.,the intracellular cleavage domain is cleaved after the capture probes isintroduced into the cell). For example, the linkage between the capturedomain, spatial barcode, and/or UMI and the cell-penetrating agent canbe a disulfide bond that is cleaved by the reducing conditions in thecell, for example, when the intracellular cleavage domain comprises adisulfide bond. Any other suitable linker can be used to release orcleave the intracellular cleavage domain of the capture probe.

(V) Positive or Neutral Oligo-Conjugated Polymers

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can be coupled to a glycol chitosanderivative. In some embodiments, the glycol chitosan derivative can becoupled with two or more molecules (e.g., nucleic acid molecules) havinga barcode (e.g., a spatial barcode). In some embodiments, the glycolchitosan derivative can be coupled with about 3, about 4, about 5, about6, about 7, about 8, about 9, about 10 or more molecules. The glycolchitosan derivative (e.g., glycol chitosan-cholesterol) can serve as ahydrophobic anchor (see Wang et al. J. Mater. Chem. B., 30:6165 (2015),which is herein incorporated by reference in its entirety). Non-limitingexamples of chitosan derivatives that can be coupled to a molecule(e.g., a nucleic acid molecule) having a barcode (e.g., a spatialbarcode) can be found in Cheung et al., Marine Drugs, 13(8): 5156-5186(2015), which is herein incorporated by reference in its entirety.

(VI) Bifunctional NHS Linker Cell-Tagging

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can be coupled to a bifunctional NHS linker.In some embodiments, the coupled bifunctional NHS linker (e.g.,bifunctional linker and the molecule having a barcode) can facilitatethe attachment of the spatial barcode to the surface of the cell. Insome embodiments, after facilitating attachment to the surface of thecell, excess NHS linker can be removed (e.g., washed away). In someembodiments, the process of coupling the molecule having a barcode canbe performed under non-anhydrous conditions to maintain the activity ofunreacted bifunctional NHS. In some embodiments, the non-anhydrouscondition can be in the presence of DMSO. In some embodiments, thenon-anhydrous condition can be in the presence of DMF.

(VII) Antibody-Tagged Primers

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can be coupled to an antibody or antigenbinding fragment thereof in a manner that facilitates attachment of themolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) to the surface of a cell. In some embodiments,facilitating attachment to the cell surface facilitates introduction ofthe molecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) into the cell. In some embodiments, the molecule (e.g.,a nucleic acid molecule) having a barcode (e.g., a spatial barcode) canbe coupled to an antibody that is directed to an antigen that is presenton the surface of a cell. In some embodiments, the molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) can becoupled to an antibody that is directed to an antigen that is present onthe surface of a plurality of cells (e.g., a plurality of cells in abiological sample). In some embodiments, the molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) can be coupledto an antibody that is directed to an antigen that is present on thesurface of a cell, a plurality of cells, or substantially all the cellspresent in a biological sample. In some embodiments, thebarcoded-antibody is directed to an intracellular antigen. Any of theexemplary methods described herein of attaching a molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) toanother molecule (e.g., an antibody or antigen fragment thereof) can beused.

(VIII) Streptavidin-Conjugated Oligonucleotides

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can attach to the surface of a cell usingbiotin-streptavidin. In some embodiments, primary amines in the sidechain of lysine residues of cell surface polypeptides are labelled withNHS-activated biotin reagents. For example, the N-terminus of apolypeptide can react with NHS-activated biotin reagents to form stableamide bonds. In some embodiments, cell-tagging agents include molecules(e.g., a nucleic acid molecule) having barcodes (e.g., a spatialbarcode) conjugated to streptavidin. In some cases, streptavidin can beconjugated to the molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) using click chemistry (e.g., maleimidemodification) as described herein. In some embodiments, a cellcontaining NHS-activated biotin incorporated into lysine side chains ofa cell surface protein forms a non-covalent bond with the streptavidinconjugated to the molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode). In some embodiments, the molecule(e.g., a nucleic acid molecule) having a barcode (e.g., a spatialbarcode) conjugated to streptavidin is itself part of a cell-taggingagent.

(IX) Dye-Tagged Oligonucleotides

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) is directly linked to a detectable label. Insome embodiments, the detectable label is any of the detectable labelsdescribed herein. In some embodiments the detectable label is afluorescent tag. In some embodiments, the physical properties of thefluorescent tags (e.g., a fluorescent tag having hydrophobic properties)can overcome the hydrophilic nature of the molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode). For example,in some embodiments, wherein the molecule is a nucleic acid molecule, afluorescent tag (e.g., BODIPY, Cy3, Atto 647N, and Rhodamine Red C2) canbe coupled to a 5′ end of the nucleic acid molecule having a barcode(e.g., a spatial barcode). In some embodiments, wherein the molecule isa nucleic acid molecule, any fluorescent tag having hydrophobicproperties can be coupled to the nucleic acid molecule having a barcode(e.g., a spatial barcode) in a manner that overcomes the hydrophilicnature of the nucleic acid molecule. Non-limiting examples offluorescent tags with hydrophobic properties include BODIPY, Cy3, Atto647N, and Rhodamine Red C2.

(X) Click-Chemistry

In some embodiments of any of the spatial analysis methods describedherein, molecules (e.g., a nucleic acid molecule) having barcodes (e.g.,a spatial barcode) are coupled to click-chemistry moieties. As usedherein, the term “click chemistry,” generally refers to reactions thatare modular, wide in scope, give high yields, generate byproducts, suchas those that can be removed by nonchromatographic methods, and arestereospecific (but not necessarily enantioselective) (see, e.g., Angew.Chem. Int. Ed., 2001, 40(11):2004-2021, which is incorporated herein byreference in its entirety). In some cases, click chemistry can describepairs of functional groups that can selectively react with each other inmild, aqueous conditions.

An example of a click chemistry reaction is the Huisgen 1,3-dipolarcycloaddition of an azide and an alkyne, i.e., copper-catalyzed reactionof an azide with an alkyne to form the 5-membered heteroatom ring1,2,3-triazole. The reaction is also known as a Cu(I)-CatalyzedAzide-Alkyne Cycloaddition (CuAAC), a Cu(I) click chemistry or a Cu+click chemistry. Catalysts for the click chemistry include, but are notlimited to, Cu(I) salts, or Cu(I) salts made in situ by reducing Cu(II)reagents to Cu(I) reagents with a reducing reagent (Pharm Res. 2008,25(10): 2216-2230, which is incorporated herein by reference in itsentirety). Known Cu(II) reagents for the click chemistry can include,but are not limited to, the Cu(II)-(TBTA) complex and the Cu(II) (THPTA)complex. TBTA, which istris-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, also known astris-(benzyltriazolylmethyl)amine, can be a stabilizing ligand for Cu(I)salts. THPTA, which is tris-(hydroxypropyltriazolylmethyl)amine, isanother example of a stabilizing agent for Cu(I). Other conditions canalso be used to construct the 1,2,3-triazole ring from an azide and analkyne using copper-free click chemistry, such as the Strain-promotedAzide- Alkyne Click chemistry reaction (SPAAC) (see, e.g., Chem.Commun., 2011, 47:6257-6259 and Nature, 2015, 519(7544):486-90, each ofwhich is incorporated herein by reference in its entirety).

(XI) Receptor-Ligand Systems

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can be coupled to a ligand, wherein the ligandis part of a receptor-ligand interaction on the surface of a cell. Forexample, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can be coupled to a ligand that interactsselectively with a cell surface receptor thereby targeting the molecule(e.g., a nucleic acid molecule) having a barcode (e.g., a spatialbarcode) to a specific cell. Non-limiting examples of receptor-ligandsystems that can be used include integrin receptor-ligand interactions,GPCR receptor-ligand interactions, RTK receptor-ligand interactions, andTLR-ligand interactions (see Juliano, Nucleic Acids Res., 44(14):6518-6548 (2016), which is incorporated herein by reference in itsentirety). Any of the methods described herein for attaching a molecule(e.g., a nucleic acid molecule) having a barcode (e.g., a spatialbarcode) to a ligand (e.g., any of the methods described herein relatingto attaching a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) to an antibody) can be used.

(XII) Covalent Binding Systems via Amine or Thiol Functionalities

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can incorporate reactive functional groups atsites within the molecule (e.g., with a nucleic acid sequence). In suchcases, the reactive functional groups can facilitate conjugation toligands and/or surfaces. In some embodiments, a molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) caninclude thiol modifiers that are designed to react with a broad array ofactivated accepting groups (e.g., maleimide and gold microspheres). Forexample, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) having thiol modifiers can interact with amaleimide-conjugated peptide thereby resulting in labelling of thepeptide. In some embodiments, maleimide-conjugated peptides are presenton the surface of a cell whereupon interaction with the thiol-modifiedmolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode), the molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) is coupled to the surface of the cell.Non-limiting examples of thiol modifiers include: 5′ thiol modifier C6S—S, 3′ thiol modifier C3 S—S, dithiol, 3′thiol modifier oxa 6—S—S, anddithiol serinol.

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can include amine modifiers, e.g., aminemodifiers that are designed to attach to another molecule in thepresence of an acylating agent. In some embodiments, a molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) caninclude amine modifiers that are designed to attach to a broad array oflinkage groups (e.g., carbonyl amide, thiourea, sulfonamide, andcarboxamide). For example, a molecule (e.g., a nucleic acid molecule)having a barcode (e.g., a spatial barcode) and an amine modifier caninteract with a sulfonamide-conjugated peptide thereby resulting inlabelling of the peptide. In some embodiments, sulfonamide-conjugatedpeptides are present on the surface of a cell whereupon interaction withthe amine-modified molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode), the molecule (e.g., a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) is coupled to thesurface of the cell. Non-limiting example of amine modifiers include:DMS(O)MT-Amino-Modifier-C6, Amino-Modifier-C3-TFA, Amino-Modifier-C12,Amino-Modifier-C6-TFA, Amino-dT, Amino-Modifier-5, Amino-Modifier-C2-dT,Amino-Modifier-C6-dT, and 3′-Amino-Modifier-C7.

As another example, a molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) can incorporate reactive functionalgroups at sites within the molecule (e.g., with a nucleic acid sequence)such as N-hydroxysuccinimide (NHS). In some embodiments, amines (e.g.,amine-containing peptides) are present on the surface of a cellwhereupon interaction with the NHS-modified molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode), the molecule(e.g., a nucleic acid molecule) having a barcode (e.g., a spatialbarcode) is coupled to the surface of the cell. In some embodiments, amolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) is reacted with a bifunctional NHS linker to form anNHS-modified molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode).

In some embodiments, a molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) can be coupled to a biocompatibleanchor for cell membrane (BAM). For example, a BAM can include moleculesthat comprise an oleyl group and PEG. The oleyl group can facilitateanchoring the molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) to a cell, and the PEG can increase watersolubility. In some embodiments, oleyl-PEG-NHS can be coupled to amolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) using NHS chemistry.

(XIII) Azide-Based Systems

In some embodiments, wherein the molecule (e.g., with a nucleic acidsequence) incorporates reactive functional groups at sites within themolecule, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can be coupled to an azide group on a cellsurface. In some embodiments, the reactive functional group is analkynyl group. In some embodiments, click chemistry as described hereincan be used to attach the alkynyl-modified molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) to an azidegroup on the cell surface. An azide group can be attached to the cellsurface through a variety of methods. For example, NHS chemistry can beused to attach an azide group to the cell surface. In some embodiments,N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz), which contains anazide group, can react with sialic acid on the surface of a cell toattach azide to the cell surface. In some embodiments, azide is attachedto the cell surface by bio-orthogonal expression of azide. For example,azide is incubated with the cells. In some embodiments, thealkynyl-modified molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) can attach to the surface of a cellvia an azide group in the presence of copper. In some embodiments, thealkynyl-modified molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) can attach to the surface of a cellvia an azide group in the absence of copper.

(XIV) Lectin-Based Systems

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can be coupled to a lectin that facilitatesattachment of the molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) to a cell surface. Lectin can bind toglycans, e.g., glycans on the surface of cells. In some embodiments, themolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) has an incorporated reactive functional group such asan azide group. In some embodiments, the molecule (e.g., a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) and an azide groupis reacted with a modified lectin, e.g., a lectin modified using NHSchemistry to introduce an azide reactive group. In some embodiments, alive cell is labelled with a lectin-modified molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode). In someembodiments, a fixed cell is labelled with a lectin-modified molecule(e.g., a nucleic acid molecule) having a barcode (e.g., a spatialbarcode). In some embodiments, a permeabilized cell is labelled with alectin-modified molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode). In some embodiments, organelles inthe secretory pathway can be labelled with a lectin-modified molecule(e.g., a nucleic acid molecule) having a barcode (e.g., a spatialbarcode).

(B) Methods for Separating a Sample Into Single Cells or Cell Groups

Some embodiments of any of the methods described herein can includeseparating a biological sample into single cells, cell groups, types ofcells, or a region or regions of interest. For example, a biologicalsample can be separated into single cells, cell groups, types of cells,or a region or regions of interest before being contacted with one ormore capture probes. In other examples, a biological sample is firstcontacted with one or more capture probes, and then separated intosingle cells, cell groups, types of cells, or a region or regions ofinterest.

In some embodiments, a biological sample can be separated into chucksusing pixelation. Pixelation can include the steps of providing abiological sample, and punching out one or more portions of thebiological sample. The punched out portions of the biological sample canthen be used to perform any of the methods described herein. In someembodiments, the punched-out portions of the biological sample can be ina random pattern or a designed pattern. In some embodiments, thepunched-out portions of the biological sample can be focused on a regionof interest or a subcellular structure in the biological sample.

FIG. 20A is a workflow schematic illustrating exemplary, non-limiting,non-exhaustive steps for “pixelating” a sample, wherein the sample iscut, stamped, microdissected, or transferred by hollow-needle ormicroneedle, moving a small portion of the sample into an individualpartition or well.

FIG. 20B is a schematic depicting multi-needle pixelation, wherein anarray of needles is punched through a sample on a scaffold, intonanowells containing beads (e.g., gel beads) and reagents. Once theneedle is in the nanowell, the cell(s) are ejected.

In some embodiments, a biological sample (e.g., a tissue sample ortissue section) is divided into smaller portions as compared to theoriginal biological sample size (“chunks”) before performance of any ofthe spatial analysis methods described herein. In some embodiments, themethods can include spatial barcoding of FFPE “chunks” via barcodesapplied in a spatially well-defined pattern (e.g., array printing). Inorder to associate a spatial barcode with a particular “chunk” ofbiological sample, the barcode (e.g., a spatial barcode) can be of asufficient length to prevent diffusion of the barcode in subsequentsteps, or the spatial barcode can be covalently applied to the FFPEsample. In some embodiments, the spatial barcode is unique to each FFPEchunk. In some embodiments, spatial barcodes can be embedded onto anFFPE slide (e.g., within a matrix, such as a wax or a hydrogel). In someembodiments, the FFPE slide is heated (e.g., wax is heated) prior toaddition of the spatial barcodes. In some embodiments, after addition ofthe spatial barcodes, the FFPE slide can be cooled and cut ordissociated into chunks. Methods of chunking (e.g., cutting) biologicalsamples are known in the art. For example, in a non-limiting example,chunking of biological samples can be done in various ways such as lasermicrodissection, mechanical means, acoustic (e.g., sonication) means, orany other method described herein. In some embodiments,fluorophores/Qdots, etc. can be embedded in the chunk to preservespatial information about the biological sample. Barcoding at this stepenables massively parallel encapsulation of chunks while retaining localspatial information (e.g., tumor versus normal/healthy cells). In someembodiments, chunking of a biological sample (e.g., a tissue section)can result in single-cell chunks of the biological sample. In otherembodiments, chunking of a biological sample can be performed to obtainchunks that correspond to diseased portions of the biological sample. Inanother embodiment, chunking of biological samples can be performed toobtain discrete chunks of the biological sample that correspond todiseased or healthy portions of the biological sample. In someembodiments, chunking of biological samples can be performed to obtainchunks that correspond to specific cell types (e.g., chunking based onfluorescent or chemiluminescent imaging of antibodies bound to targetproteins) in the biological sample.

In some embodiments, the spatially-barcoded chunks can be furtherprocessed. For example, the spatially-barcoded chunk can be individuallyencapsulated (e.g., a matrix, emulsion, or hydrogel). In someembodiments, the spatially-barcoded chunk can be encapsulated in apartition (e.g., a well, droplet, channel, or vesicle). In someembodiments, the spatially-barcoded chunk can be encapsulated in avesicle. In some embodiments, the vesicle can comprise a lipid bilayer.In some embodiments, the spatially-barcoded FFPE chunk can beencapsulated with a uniquely barcoded bead. In some embodiments, theuniquely barcoded bead can have a functional domain, a cleavage domain,a unique molecular identifier, and a capture domain, or combinationsthereof. In some embodiments, the encapsulated spatially-barcoded FFPEchunk and the uniquely barcoded bead can be heated to deparaffinize theFFPE sample. In some embodiments, the encapsulated spatially-barcodedFFPE chunk and the uniquely barcoded bead can be treated with xylene todeparaffinize the FFPE sample. In some embodiments, the deparaffinizedsample can be treated to de-crosslink methylene bridges in a singlestep. In some embodiments, additional steps can be performed when, forexample, de-crosslinking chemistry is incompatible with barcoding orlibrary preparation steps. In some embodiments, after de-crosslinkingmethylene bridges, the nucleic acids originating or present in the chunkcan bind to the uniquely barcoded bead. In some embodiments, after thespatial barcode binds the uniquely barcoded bead, the encapsulation canbe disrupted (e.g., lysed, melted, or removed) and the barcoded beadscan be collected. In some embodiments, the collected barcoded beads canbe washed and re-encapsulated. In some embodiments, the nucleic acidsassociated with the bead (e.g., spatial barcode, unique barcode, analytetranscript) can be amplified (e.g., PCR amplified) and processed (e.g.,sequenced) according to any of the methods described herein.

In some embodiments, a biological sample can be divided or portionedusing laser capture microdissection (e.g., highly-multiplexed lasercapture microdissection).

(C) Release and Amplification of Analytes

In some embodiments, lysis reagents can be added to the sample tofacilitate release of analyte(s) from a sample. Examples of lysis agentsinclude, but are not limited to, bioactive reagents such as lysisenzymes that are used for lysis of different cell types, e.g., grampositive or negative bacteria, plants, yeast, mammalian, such aslysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase,and a variety of other commercially available lysis enzymes. Other lysisagents can additionally or alternatively be co-partitioned with thebiological sample to cause the release of the sample’s contents into thepartitions. In some embodiments, surfactant-based lysis solutions can beused to lyse cells, although these can be less desirable foremulsion-based systems where the surfactants can interfere with stableemulsions. Lysis solutions can include ionic surfactants such as, forexample, sarcosyl and sodium dodecyl sulfate (SDS). Electroporation,thermal, acoustic or mechanical cellular disruption can also be used incertain embodiments, e.g., non-emulsion based partitioning such asencapsulation of biological materials that can be in addition to or inplace of droplet partitioning, where any pore volume of the encapsulateis sufficiently small to retain nucleic acid fragments of a given size,following cellular disruption.

In addition to the permeabilization agents, other reagents can also beadded to interact with the biological sample, including, for example,DNase and RNase inactivating agents or inhibitors or chelating agents,such as EDTA, and other reagents to allow for subsequent processing ofanalytes from the sample. In other embodiments, nucleases, such as DNaseor RNAse, or proteases, such as pepsin or proteinase K, are added to thesample.

Further reagents that can be added to a sample, include, for example,endonucleases to fragment DNA, DNA polymerase enzymes, and dNTPs used toamplify nucleic acids. Other enzymes that can also be added to thesample include, but are not limited to, polymerase, transposase, ligase,proteinase K, and DNAse, etc. Additional reagents can also includereverse transcriptase enzymes, including enzymes with terminaltransferase activity, primers, and switch oligonucleotides. In someembodiments, template switching can be used to increase the length of acDNA, e.g., by appending a predefined nucleic acid sequence to the cDNA.

If a tissue sample is not permeabilized sufficiently, the amount ofanalyte captured on the substrate can be too low to enable adequateanalysis. Conversely, if the tissue sample is too permeable, the analytecan diffuse away from its origin in the tissue sample, such that therelative spatial relationship of the analytes within the tissue sampleis lost. Hence, a balance between permeabilizing the tissue sampleenough to obtain good signal intensity while still maintaining thespatial resolution of the analyte distribution in the tissue sample isdesired.

In some embodiments, where the biological sample includes live cells,permeabilization conditions can be modified so that the live cellsexperience only brief permeabilization (e.g., through short repetitivebursts of electric field application), thereby allowing one or moreanalytes to migrate from the live cells to the substrate while retainingcellular viability.

In some embodiments, after contacting a biological sample with asubstrate that include capture probes, a removal step is performed toremove all or a portion of the biological sample from the substrate. Insome embodiments, the removal step includes enzymatic or chemicaldegradation of the permeabilized cells of the biological sample. Forexample, the removal step can include treating the biological sampleswith an enzyme (e.g., proteinase K) to remove at least a portion of thebiological sample from the first substrates. In some embodiments, theremoval step can include ablation of the tissue (e.g., laser ablation).

In some embodiments, where RNA is captured from cells in a sample, oneor more RNA species of interest can be selectively enriched. Forexample, one or more species of RNA of interest can be selected byaddition of one or more oligonucleotides. One or more species of RNA canbe selectively down-selected (e.g., removed) using any of a variety ofmethods. For example, probes can be administered to a sample thatselectively hybridize to ribosomal RNA (rRNA), thereby reducing the pooland concentration of rRNA in the sample. Subsequent application of thecapture probes to the sample can result in improved RNA capture due tothe reduction in non-specific RNA present in the sample. In someembodiments, the additional oligonucleotide is a sequence used forpriming a reaction by a polymerase. For example, one or more primersequences with sequence complementarity to one or more RNAs of interest,can be used to amplify the one or more RNAs of interest, therebyselectively enriching these RNAs. In some embodiments, anoligonucleotide with sequence complementarity to the complementarystrand of captured RNA (e.g., cDNA) can bind to the cDNA. In onenon-limiting example, biotinylated oligonucleotides with sequencecomplementary to one or more cDNA of interest binds to the cDNA and canbe selected using biotinylation-strepavidin affinity in any number ofmethods known to the field (e.g., streptavidin beads).

In some embodiments, any of the spatial analysis methods describedherein can include modulating the rate of interaction between biologicalanalytes from the biological sample and the capture probes on the array.In some embodiments, modulating the rate of interaction can occur bymodulating the biological sample (e.g., modulating temperature or pH).In some embodiments, modulating the rate of interaction includes usingexternal stimuli. Non-limiting examples of external stimuli that can beused to modulate the rate of interaction include light, temperature,small molecules, enzymes, and/or an activating reagent. In one example,light can be used to activate a polymerase in a nucleic acid extensionreaction. In another example, temperature can be used to modulatehybridization between two complementary nucleic acid molecules.

Nucleic acid analytes can be amplified using a polymerase chain reaction(e.g., digital PCR, quantitative PCR, or real time PCR), isothermalamplification, or any nucleic acid amplification or extension reactionsdescribed herein, or known in the art.

(D) Partitioning

As discussed above, in some embodiments, the sample can optionally beseparated into single cells, cell groups (e.g., based on cell sub-typeor gene expression profile), or other fragments/pieces that are smallerthan the original sample. Each of these smaller portions of the samplecan be analyzed to obtain spatially-resolved analyte information fromthe sample. Non-limiting partitioning methods are described herein.

For samples that have been separated into smaller fragments - andparticularly, for samples that have been disaggregated, dissociated, orotherwise separated into individual cells - one method for analyzing thefragments involves partitioning the fragments into individual partitions(e.g., fluid droplets), and then analyzing the contents of thepartitions. In general, each partition maintains separation of its owncontents from the contents of other partitions. For example, thepartition can be a droplet in an emulsion.

The methods described herein provide for the compartmentalization orpartitioning of a cell (e.g., a cell) from a sample into discretecompartments or voxels. As used herein, each “voxel” represents a3-dimensional volumetric unit. In some embodiments, a voxel maintainsseparation of its own contents from the contents of other voxels. Avoxel can be one partition of an array of partitions of volume. Forexample, a voxel can be one partition of an array of discrete partitionsinto which a 3-dimensional object is divided. As another example,members of a plurality of photo-crosslinkable polymer precursors can becross-linked into voxels that are part of an array of thephoto-crosslinked polymer covering the substrate or a portion of thesubstrate. Unique identifiers, e.g., barcodes, may be previously,subsequently, or concurrently delivered to the cell, in order to allowfor the later attribution of the characteristics of the cell to theparticular voxel. In some embodiments, a voxel has defined dimensions.In some embodiments, a voxel comprises a single cell.

For example, a substrate can be coated with a DTT-sensitive hydrogel andthen contacted with a biological sample. Optionally, capture probesattached to the substrate are released from the substrate such that thereleased capture probes are introduced into the biological sample and atleast one released capture probe interacts with at least one biologicalanalyte present in the biological sample via the capture domain. Thebiological sample and substrate can be assembled into a flow-cell and aphoto-crosslinkable polymer precursor added. The cells of the biologicalsample can be then crosslinked into hydrogel-voxels of defineddimensions using a light source. The flow-cell can be dismantled andwashed to remove unpolymerized polymer precursors. The coating can betreated with DTT to yield single-cell partitions for use in downstreamapplications. The capture probes/biological analytes can be analyzed,and the spatial information of the spatially-barcoded features can beused to determine the spatial location of the captured biologicalanalytes in the biological sample.

In addition to analytes, a partition can include additional components,and in particular, one or more beads. A partition can include a singlegel bead, a single cell bead, or both a single cell bead and single gelbead.

A partition can also include one or more reagents. Unique identifiers,such as barcodes, can be injected into the droplets previous to,subsequent to, or concurrently with droplet generation, such as via amicrocapsule (e.g., bead). Microfluidic channel networks (e.g., on achip) can be utilized to generate partitions. Alternative mechanisms canalso be employed in the partitioning of individual biological particles,including porous membranes through which aqueous mixtures of cells areextruded into non-aqueous fluids.

The partitions can be flowable within fluid streams. The partitions caninclude, for example, micro-vesicles that have an outer barriersurrounding an inner fluid center or core. In some cases, the partitionscan include a porous matrix that is capable of entraining and/orretaining materials within its matrix. The partitions can be droplets ofa first phase within a second phase, wherein the first and second phasesare immiscible. For example, the partitions can be droplets of aqueousfluid within a non-aqueous continuous phase (e.g., oil phase). Inanother example, the partitions can be droplets of a non-aqueous fluidwithin an aqueous phase. In some examples, the partitions can beprovided in a water-in-oil emulsion or oil-in-water emulsion. A varietyof different vessels are described in, for example, U.S. Pat.Application Publication No. 2014/0155295, the entire contents of whichare incorporated herein by reference. Emulsion systems for creatingstable droplets in non-aqueous or oil continuous phases are described,for example, in U.S. Pat. Application Publication No. 2010/0105112, theentire contents of which are incorporated herein by reference.

For droplets in an emulsion, allocating individual particles to discretepartitions can be accomplished, for example, by introducing a flowingstream of particles in an aqueous fluid into a flowing stream of anon-aqueous fluid, such that droplets are generated at the junction ofthe two streams. Fluid properties (e.g., fluid flow rates, fluidviscosities, etc.), particle properties (e.g., volume fraction, particlesize, particle concentration, etc.), microfluidic architectures (e.g.,channel geometry, etc.), and other parameters can be adjusted to controlthe occupancy of the resulting partitions (e.g., number of analytes perpartition, number of beads per partition, etc.) For example, partitionoccupancy can be controlled by providing the aqueous stream at a certainconcentration and/or flow rate of analytes.

To generate single analyte partitions, the relative flow rates of theimmiscible fluids can be selected such that, on average, the partitionscan contain less than one analyte per partition to ensure that thosepartitions that are occupied are primarily singly occupied. In somecases, partitions among a plurality of partitions can contain at mostone analyte. In some embodiments, the various parameters (e.g., fluidproperties, particle properties, microfluidic architectures, etc.) canbe selected or adjusted such that a majority of partitions are occupied,for example, allowing for only a small percentage of unoccupiedpartitions. The flows and channel architectures can be controlled as toensure a given number of singly occupied partitions, less than a certainlevel of unoccupied partitions and/or less than a certain level ofmultiply occupied partitions.

The channel segments described herein can be coupled to any of a varietyof different fluid sources or receiving components, includingreservoirs, tubing, manifolds, or fluidic components of other systems.As will be appreciated, the microfluidic channel structure can have avariety of geometries. For example, a microfluidic channel structure canhave one or more than one channel junction. As another example, amicrofluidic channel structure can have 2, 3, 4, or 5 channel segmentseach carrying particles that meet at a channel junction. Fluid can bedirected to flow along one or more channels or reservoirs via one ormore fluid flow units. A fluid flow unit can include compressors (e.g.,providing positive pressure), pumps (e.g., providing negative pressure),actuators, and the like to control flow of the fluid. Fluid can also orotherwise be controlled via applied pressure differentials, centrifugalforce, electrokinetic pumping, vacuum, capillary, and/or gravity flow.

A partition can include one or more unique identifiers, such asbarcodes. Barcodes can be previously, subsequently, or concurrentlydelivered to the partitions that hold the compartmentalized orpartitioned biological particle. For example, barcodes can be injectedinto droplets previous to, subsequent to, or concurrently with dropletgeneration. The delivery of the barcodes to a particular partitionallows for the later attribution of the characteristics of theindividual biological particle to the particular partition. Barcodes canbe delivered, for example on a nucleic acid molecule (e.g., anoligonucleotide), to a partition via any suitable mechanism. Barcodednucleic acid molecules can be delivered to a partition via amicrocapsule. A microcapsule, in some instances, can include a bead.

In some embodiments, barcoded nucleic acid molecules can be initiallyassociated with the microcapsule and then released from themicrocapsule. Release of the barcoded nucleic acid molecules can bepassive (e.g., by diffusion out of the microcapsule). In addition oralternatively, release from the microcapsule can be upon application ofa stimulus which allows the barcoded nucleic acid nucleic acid moleculesto dissociate or to be released from the microcapsule. Such stimulus candisrupt the microcapsule, an interaction that couples the barcodednucleic acid molecules to or within the microcapsule, or both. Suchstimulus can include, for example, a thermal stimulus, photo-stimulus,chemical stimulus (e.g., change in pH or use of a reducing agent(s)), amechanical stimulus, a radiation stimulus; a biological stimulus (e.g.,enzyme), or any combination thereof.

In some embodiments, one more barcodes (e.g., spatial barcodes, UMIs, ora combination thereof) can be introduced into a partition as part of theanalyte. As described previously, barcodes can be bound to the analytedirectly, or can form part of a capture probe or analyte capture agentthat is hybridized to, conjugated to, or otherwise associated with ananalyte, such that when the analyte is introduced into the partition,the barcode(s) are introduced as well.

FIG. 21 depicts an exemplary workflow, where a sample is contacted witha spatially-barcoded capture probe array and the sample is fixed,stained, and imaged 2101, as described elsewhere herein. The captureprobes can be cleaved from the array 2102 using any method as describedherein. The capture probes can diffuse toward the cells by eitherpassive or active migration as described elsewhere herein. The captureprobes may then be introduced to the sample 2103 as described elsewhereherein, wherein the capture probe is able to gain entry into the cell inthe absence of cell permeabilization, using one of the cell penetratingpeptides or lipid delivery systems described herein. The sample can thenbe optionally imaged in order to confirm probe uptake, via a reportermolecule incorporated within the capture probe 2104. The sample can thenbe separated from the array and undergo dissociation 2105, wherein thesample is separated into single cells or small groups of cells. Once thesample is dissociated, the single cells can be introduced to an oil-inwater droplet 2106, wherein a single cell is combined with reagentswithin the droplet and processed so that the spatial barcode thatpenetrated the cell labels the contents of that cell within the droplet.Other cells undergo separately partitioned reactions concurrently. Thecontents of the droplet is then sequenced 2107 in order to associate aparticular cell or cells with a particular spatial location within thesample 2108.

As described above, FIG. 16 shows an example of a microfluidic channelstructure for partitioning individual analytes (e.g., cells) intodiscrete partitions. FIGS. 17A and 17C also show other examples ofmicrofluidic channel structures that can be used for delivering beads todroplets.

A variety of different beads can be incorporated into partitions asdescribed above. In some embodiments, for example, non-barcoded beadscan be incorporated into the partitions. For example, where thebiological particle (e.g., a cell) that is incorporated into thepartitions carries one or more barcodes (e.g., spatial barcode(s),UMI(s), and combinations thereof), the bead can be a non-barcoded bead.

In some embodiments, a barcode carrying bead can be incorporated intopartitions. For example, a nucleic acid molecule, such as anoligonucleotide, can be coupled to a bead by a releasable linkage, suchas, for example, a disulfide linker. The same bead can be coupled (e.g.,via releasable linkage) to one or more other nucleic acid molecules. Thenucleic acid molecule can be or include a barcode. As noted elsewhereherein, the structure of the barcode can include a number of sequenceelements.

The nucleic acid molecule can include a functional domain that can beused in subsequent processing. For example, the functional domain caninclude one or more of a sequencer specific flow cell attachmentsequence (e.g., a P5 sequence for Illumina® sequencing systems) and asequencing primer sequence (e.g., a R1 primer for Illumina® sequencingsystems). The nucleic acid molecule can include a barcode sequence foruse in barcoding the sample (e.g., DNA, RNA, protein, etc.). In somecases, the barcode sequence can be bead-specific such that the barcodesequence is common to all nucleic acid molecules coupled to the samebead. Alternatively or in addition, the barcode sequence can bepartition-specific such that the barcode sequence is common to allnucleic acid molecules coupled to one or more beads that are partitionedinto the same partition. The nucleic acid molecule can include aspecific priming sequence, such as an mRNA specific priming sequence(e.g., poly (T) sequence), a targeted priming sequence, and/or a randompriming sequence. The nucleic acid molecule can include an anchoringsequence to ensure that the specific priming sequence hybridizes at thesequence end (e.g., of the mRNA). For example, the anchoring sequencecan include a random short sequence of nucleotides, such as a 1-mer,2-mer, 3-mer or longer sequence, which can ensure that a poly(T) segmentis more likely to hybridize at the sequence end of the poly(A) tail ofthe mRNA.

The nucleic acid molecule can include a unique molecular identifyingsequence (e.g., unique molecular identifier (UMI)). In some embodiments,the unique molecular identifying sequence can include from about 5 toabout 8 nucleotides. Alternatively, the unique molecular identifyingsequence can include less than about 5 or more than about 8 nucleotides.The unique molecular identifying sequence can be a unique sequence thatvaries across individual nucleic acid molecules coupled to a singlebead.

In some embodiments, the unique molecular identifying sequence can be arandom sequence (e.g., such as a random N-mer sequence). For example,the UMI can provide a unique identifier of the starting mRNA moleculethat was captured, in order to allow quantitation of the number oforiginal expressed RNA.

In general, an individual bead can be coupled to any number ofindividual nucleic acid molecules, for example, from one to tens tohundreds of thousands or even millions of individual nucleic acidmolecules. The respective barcodes for the individual nucleic acidmolecules can include both common sequence segments or relatively commonsequence segments and variable or unique sequence segments betweendifferent individual nucleic acid molecules coupled to the same bead.

Within any given partition, all of the cDNA transcripts of theindividual mRNA molecules can include a common barcode sequence segment.However, the transcripts made from the different mRNA molecules within agiven partition can vary at the unique molecular identifying sequencesegment (e.g., UMI segment). Beneficially, even following any subsequentamplification of the contents of a given partition, the number ofdifferent UMIs can be indicative of the quantity of mRNA originatingfrom a given partition. As noted above, the transcripts can beamplified, cleaned up, and sequenced to identify the sequence of thecDNA transcript of the mRNA, as well as to sequence the barcode segmentand the UMI segment. While a poly(T) primer sequence is described, othertargeted or random priming sequences can also be used in priming thereverse transcription reaction. Likewise, although described asreleasing the barcoded oligonucleotides into the partition, in somecases, the nucleic acid molecules bound to the bead can be used tohybridize and capture the mRNA on the solid phase of the bead, forexample, in order to facilitate the separation of the RNA from othercell contents.

In some embodiments, precursors that include a functional group that isreactive or capable of being activated such that it becomes reactive canbe polymerized with other precursors to generate gel beads that includethe activated or activatable functional group. The functional group canthen be used to attach additional species (e.g., disulfide linkers,primers, other oligonucleotides, etc.) to the gel beads. For example,some precursors featuring a carboxylic acid (COOH) group canco-polymerize with other precursors to form a bead that also includes aCOOH functional group. In some cases, acrylic acid (a species comprisingfree COOH groups), acrylamide, and bis(acryloyl)cystamine can becopolymerized together to generate a bead with free COOH groups. TheCOOH groups of the bead can be activated (e.g., via1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-Hydroxysuccinimide (NHS) or4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM)) such that they are reactive (e.g., reactive to amine functionalgroups where EDC/NHS or DMTMM are used for activation). The activatedCOOH groups can then react with an appropriate species (e.g., a speciescomprising an amine functional group where the carboxylic acid groupsare activated to be reactive with an amine functional group) comprisinga moiety to be linked to the bead.

In some embodiments, a degradable bead can be introduced into apartition, such that the bead degrades within the partition and anyassociated species (e.g., oligonucleotides) are released within thedroplet when the appropriate stimulus is applied. The free species(e.g., oligonucleotides, nucleic acid molecules) can interact with otherreagents contained in the partition. For example, a polyacrylamide beadfeaturing cystamine and linked, via a disulfide bond, to a barcodesequence, can be combined with a reducing agent within a droplet of awater-in-oil emulsion. Within the droplet, the reducing agent can breakthe various disulfide bonds, resulting in bead degradation and releaseof the barcode sequence into the aqueous, inner environment of thedroplet. In another example, heating of a droplet with a bead-boundbarcode sequence in basic solution can also result in bead degradationand release of the attached barcode sequence into the aqueous, innerenvironment of the droplet.

Any suitable number of species (e.g., primer, barcoded oligonucleotide)can be associated with a bead such that, upon release from the bead, thespecies (e.g., primer, e.g., barcoded oligonucleotide) are present inthe partition at a pre-defined concentration. Such pre-definedconcentration can be selected to facilitate certain reactions forgenerating a sequencing library, e.g., amplification, within thepartition. In some cases, the pre-defined concentration of the primercan be limited by the process of producing nucleic acid molecule (e.g.,oligonucleotide) bearing beads.

A degradable bead can include one or more species with a labile bondsuch that, when the bead/species is exposed to the appropriate stimulus,the bond is broken and the bead degrades. The labile bond can be achemical bond (e.g., covalent bond, ionic bond) or can be another typeof physical interaction (e.g., van der Waals interactions, dipole-dipoleinteractions, etc.). In some embodiments, a cross-linker used togenerate a bead can include a labile bond. Upon exposure to theappropriate conditions, the labile bond can be broken and the beaddegraded. For example, upon exposure of a polyacrylamide gel bead thatincludes cystamine cross-linkers to a reducing agent, the disulfidebonds of the cystamine can be broken and the bead degraded.

A degradable bead can be useful in more quickly releasing an attachedspecies (e.g., a nucleic acid molecule, a barcode sequence, a primer,etc.) from the bead when the appropriate stimulus is applied to the beadas compared to a bead that does not degrade. For example, for a speciesbound to an inner surface of a porous bead or in the case of anencapsulated species, the species can have greater mobility andaccessibility to other species in solution upon degradation of the bead.In some embodiments, a species can also be attached to a degradable beadvia a degradable linker (e.g., disulfide linker). The degradable linkercan respond to the same stimuli as the degradable bead or the twodegradable species can respond to different stimuli. For example, abarcode sequence can be attached, via a disulfide bond, to apolyacrylamide bead comprising cystamine. Upon exposure of thebarcoded-bead to a reducing agent, the bead degrades and the barcodesequence is released upon breakage of both the disulfide linkage betweenthe barcode sequence and the bead and the disulfide linkages of thecystamine in the bead.

As will be appreciated from the above description, while referred to asdegradation of a bead, in many embodiments, degradation can refer to thedisassociation of a bound or entrained species from a bead, both withand without structurally degrading the physical bead itself. Forexample, entrained species can be released from beads through osmoticpressure differences due to, for example, changing chemicalenvironments. By way of example, alteration of bead pore volumes due toosmotic pressure differences can generally occur without structuraldegradation of the bead itself. In some cases, an increase in porevolume due to osmotic swelling of a bead can permit the release ofentrained species within the bead. In some embodiments, osmoticshrinking of a bead can cause a bead to better retain an entrainedspecies due to pore volume contraction.

Numerous chemical triggers can be used to trigger the degradation ofbeads within partitions. Examples of these chemical changes can include,but are not limited to pH-mediated changes to the integrity of acomponent within the bead, degradation of a component of a bead viacleavage of cross-linked bonds, and depolymerization of a component of abead.

In some embodiments, a bead can be formed from materials that includedegradable chemical cross-linkers, such as BAC or cystamine. Degradationof such degradable cross-linkers can be accomplished through a number ofmechanisms. In some examples, a bead can be contacted with a chemicaldegrading agent that can induce oxidation, reduction or other chemicalchanges. For example, a chemical degrading agent can be a reducingagent, such as dithiothreitol (DTT). Additional examples of reducingagents can include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane(dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), orcombinations thereof. A reducing agent can degrade the disulfide bondsformed between gel precursors forming the bead, and thus, degrade thebead.

In certain embodiments, a change in pH of a solution, such as anincrease in pH, can trigger degradation of a bead. In other embodiments,exposure to an aqueous solution, such as water, can trigger hydrolyticdegradation, and thus degradation of the bead. In some cases, anycombination of stimuli can trigger degradation of a bead. For example, achange in pH can enable a chemical agent (e.g., DTT) to become aneffective reducing agent.

Beads can also be induced to release their contents upon the applicationof a thermal stimulus. A change in temperature can cause a variety ofchanges to a bead. For example, heat can cause a solid bead to liquefy.A change in heat can cause melting of a bead such that a portion of thebead degrades. In other cases, heat can increase the internal pressureof the bead components such that the bead ruptures or explodes. Heat canalso act upon heat-sensitive polymers used as materials to constructbeads.

In addition to beads and analytes, partitions that are formed caninclude a variety of different reagents and species. For example, whenlysis reagents are present within the partitions, the lysis reagents canfacilitate the release of analytes within the partition. Examples oflysis agents include bioactive reagents, such as lysis enzymes that areused for lysis of different cell types, e.g., gram positive or negativebacteria, plants, yeast, mammalian, etc., such as lysozymes,achromopeptidase, lysostaphin, labiase, kitalase, lyticase, and avariety of other lysis enzymes available from, e.g., Sigma-Aldrich, Inc.(St. Louis, MO), as well as other commercially available lysis enzymes.Other lysis agents can additionally or alternatively be co-partitionedto cause the release analytes into the partitions. For example, in somecases, surfactant-based lysis solutions can be used to lyse cells,although these can be less desirable for emulsion based systems wherethe surfactants can interfere with stable emulsions. In someembodiments, lysis solutions can include non-ionic surfactants such as,for example, TritonX-100 and Tween 20. In some embodiments, lysissolutions can include ionic surfactants such as, for example, sarcosyland sodium dodecyl sulfate (SDS). Electroporation, thermal, acoustic ormechanical cellular disruption can also be used in certain embodiments,e.g., non-emulsion based partitioning such as encapsulation of analytesthat can be in addition to or in place of droplet partitioning, whereany pore volume of the encapsulate is sufficiently small to retainnucleic acid fragments of a given size, following cellular disruption.

Examples of other species that can be co-partitioned with analytes inthe partitions include, but are not limited to, DNase and RNaseinactivating agents or inhibitors or chelating agents, such as EDTA, andother reagents employed in removing or otherwise reducing negativeactivity or impact of different cell lysate components on subsequentprocessing of nucleic acids. Additional reagents can also beco-partitioned, including endonucleases to fragment DNA, DNA polymeraseenzymes and dNTPs used to amplify nucleic acid fragments and to attachthe barcode molecular tags to the amplified fragments.

Additional reagents can also include reverse transcriptase enzymes,including enzymes with terminal transferase activity, primers andoligonucleotides, and switch oligonucleotides (also referred to hereinas “switch oligos” or “template switching oligonucleotides”) which canbe used for template switching. In some embodiments, template switchingcan be used to increase the length of a cDNA. Template switching can beused to append a predefined nucleic acid sequence to the cDNA. In anexample of template switching, cDNA can be generated from reversetranscription of a template, e.g., cellular mRNA, where a reversetranscriptase with terminal transferase activity can add additionalnucleotides, e.g., poly(C), to the cDNA in a template independentmanner. Switch oligos can include sequences complementary to theadditional nucleotides, e.g., poly(G). The additional nucleotides (e.g.,poly(C)) on the cDNA can hybridize to the additional nucleotides (e.g.,poly(G)) on the switch oligo, whereby the switch oligo can be used bythe reverse transcriptase as template to further extend the cDNA.

Template switching oligonucleotides can include a hybridization regionand a template region. The hybridization region can include any sequencecapable of hybridizing to the target. In some cases, the hybridizationregion includes a series of G bases to complement the overhanging Cbases at the 3′ end of a cDNA molecule. The series of G bases caninclude 1 G base, 2 G bases, 3 G bases, 4 G bases, 5 G bases, or morethan 5 G bases. The template sequence can include any sequence to beincorporated into the cDNA. In some cases, the template region includesat least 1 (e.g., at least 2, 3, 4, 5 or more) tag sequences and/orfunctional sequences. Switch oligos can include deoxyribonucleic acids;ribonucleic acids; modified nucleic acids including 2-Aminopurine,2,6-Diaminopurine (2-Amino-dA), inverted dT, 5-Methyl dC,2′-deoxyInosine, Super T (5-hydroxybutynl-2′-deoxyuridine), Super G(8-aza-7-deazaguanosine), locked nucleic acids (LNAs), unlocked nucleicacids (UNAs, e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2′Fluoro bases (e.g., Fluoro C, Fluoro U, Fluoro A, and Fluoro G), andcombinations of the foregoing.

In some embodiments, beads that are partitioned with the analyte caninclude different types of oligonucleotides bound to the bead, where thedifferent types of oligonucleotides bind to different types of analytes.For example, a bead can include one or more first oligonucleotides(which can be capture probes, for example) that can bind or hybridize toa first type of analyte, such as mRNA for example, and one or moresecond oligonucleotides (which can be capture probes, for example) thatcan bind or hybridize to a second type of analyte, such as gDNA forexample. Partitions can also include lysis agents that aid in releasingnucleic acids from the co-partitioned cell, and can also include anagent (e.g., a reducing agent) that can degrade the bead and/or breakcovalent linkages between the oligonucleotides and the bead, releasingthe oligonucleotides into the partition. The released barcodedoligonucleotides (which can also be barcoded) can hybridize with mRNAreleased from the cell and also with gDNA released from the cell.

Barcoded constructs thus formed from hybridization can include a firsttype of construct that includes a sequence corresponding to an originalbarcode sequence from the bead and a sequence corresponding to atranscript from the cell, and a second type of construct that includes asequence corresponding to the original barcode sequence from the beadand a sequence corresponding to genomic DNA from the cell. The barcodedconstructs can then be released/removed from the partition and, in someembodiments, further processed to add any additional sequences. Theresulting constructs can then be sequenced, the sequencing dataprocessed, and the results used to spatially characterize the mRNA andthe gDNA from the cell.

In another example, a partition includes a bead that includes a firsttype of oligonucleotide (e.g., a first capture probe) with a firstbarcode sequence, a poly(T) priming sequence that can hybridize with thepoly(A) tail of an mRNA transcript, and a UMI barcode sequence that canuniquely identify a given transcript. The bead also includes a secondtype of oligonucleotide (e.g., a second capture probe) with a secondbarcode sequence, a targeted priming sequence that is capable ofspecifically hybridizing with a third barcoded oligonucleotide (e.g., ananalyte capture agent) coupled to an antibody that is bound to thesurface of the partitioned cell. The third barcoded oligonucleotideincludes a UMI barcode sequence that uniquely identifies the antibody(and thus, the particular cell surface feature to which it is bound).

In this example, the first and second barcoded oligonucleotides includethe same spatial barcode sequence (e.g., the first and second barcodesequences are the same), which permits downstream association ofbarcoded nucleic acids with the partition. In some embodiments, however,the first and second barcode sequences are different.

The partition also includes lysis agents that aid in releasing nucleicacids from the cell and can also include an agent (e.g., a reducingagent) that can degrade the bead and/or break a covalent linkage betweenthe barcoded oligonucleotides and the bead, releasing them into thepartition. The first type of released barcoded oligonucleotide canhybridize with mRNA released from the cell and the second type ofreleased barcoded oligonucleotide can hybridize with the third type ofbarcoded oligonucleotide, forming barcoded constructs.

The first type of barcoded construct includes a spatial barcode sequencecorresponding to the first barcode sequence from the bead and a sequencecorresponding to the UMI barcode sequence from the first type ofoligonucleotide, which identifies cell transcripts. The second type ofbarcoded construct includes a spatial barcode sequence corresponding tothe second barcode sequence from the second type of oligonucleotide, anda UMI barcode sequence corresponding to the third type ofoligonucleotide (e.g., the analyte capture agent) and used to identifythe cell surface feature. The barcoded constructs can then bereleased/removed from the partition and, in some embodiments, furtherprocessed to add any additional sequences. The resulting constructs arethen sequenced, sequencing data processed, and the results used tocharacterize the mRNA and cell surface feature of the cell.

The foregoing discussion involves two specific examples of beads witholigonucleotides for analyzing two different analytes within apartition. More generally, beads that are partitioned can have any ofthe structures described previously, and can include any of thedescribed combinations of oligonucleotides for analysis of two or more(e.g., three or more, four or more, five or more, six or more, eight ormore, ten or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 ormore, 40 or more, 50 or more) different types of analytes within apartition. Examples of beads with combinations of different types ofoligonucleotides (e.g., capture probes) for concurrently analyzingdifferent combinations of analytes within partitions include, but arenot limited to: (a) genomic DNA and cell surface features (e.g., usingthe analyte capture agents described herein); (b) mRNA and a lineagetracing construct; (c) mRNA and cell methylation status; (d) mRNA andaccessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq); (e)mRNA and cell surface or intracellular proteins and/or metabolites; (f)a barcoded analyte capture agent (e.g., the MHC multimers describedherein) and a V(D)J sequence of an immune cell receptor (e.g., T-cellreceptor); and (g) mRNA and a perturbation agent (e.g., a CRISPRcrRNA/sgRNA, TALEN, zinc finger nuclease, and/or antisenseoligonucleotide as described herein). In some embodiments, aperturbation agent can be a small molecule, an antibody, a drug, anaptamer, a nucleic acid (e.g., miRNA), a physical environmental (e.g.,temperature change), or any other known perturbation agents.

(E) Sequencing Analysis

After analytes from the sample have hybridized or otherwise beenassociated with capture probes, analyte capture agents, or otherbarcoded oligonucleotide sequences according to any of the methodsdescribed above in connection with the general spatial cell-basedanalytical methodology, the barcoded constructs that result fromhybridization/association are analyzed via sequencing to identify theanalytes.

In some embodiments, where a sample is barcoded directly viahybridization with capture probes or analyte capture agents hybridized,bound, or associated with either the cell surface, or introduced intothe cell, as described above, sequencing can be performed on the intactsample. Alternatively, if the barcoded sample has been separated intofragments, cell groups, or individual cells, as described above,sequencing can be performed on individual fragments, cell groups, orcells. For analytes that have been barcoded via partitioning with beads,as described above, individual analytes (e.g., cells, or cellularcontents following lysis of cells) can be extracted from the partitionsby breaking the partitions, and then analyzed by sequencing to identifythe analytes.

A wide variety of different sequencing methods can be used to analyzebarcoded analyte constructs. In general, sequenced polynucleotides canbe, for example, nucleic acid molecules such as deoxyribonucleic acid(DNA) or ribonucleic acid (RNA), including variants or derivativesthereof (e.g., single stranded DNA or DNA/RNA hybrids, and nucleic acidmolecules with a nucleotide analog).

Sequencing of polynucleotides can be performed by various commercialsystems. More generally, sequencing can be performed using nucleic acidamplification, polymerase chain reaction (PCR) (e.g., digital PCR anddroplet digital PCR (ddPCR), quantitative PCR, real time PCR, multiplexPCR, PCR-based singleplex methods, emulsion PCR), and/or isothermalamplification.

Other examples of methods for sequencing genetic material include, butare not limited to, DNA hybridization methods (e.g., Southern blotting),restriction enzyme digestion methods, Sanger sequencing methods,next-generation sequencing methods (e.g., single-molecule real-timesequencing, nanopore sequencing, and Polony sequencing), ligationmethods, and microarray methods. Additional examples of sequencingmethods that can be used include targeted sequencing, single moleculereal-time sequencing, exon sequencing, electron microscopy-basedsequencing, panel sequencing, transistor-mediated sequencing, directsequencing, random shotgun sequencing, Sanger dideoxy terminationsequencing, whole-genome sequencing, sequencing by hybridization,pyrosequencing, capillary electrophoresis, gel electrophoresis, duplexsequencing, cycle sequencing, single-base extension sequencing,solid-phase sequencing, high-throughput sequencing, massively parallelsignature sequencing, co-amplification at lower denaturationtemperature-PCR (COLD-PCR), sequencing by reversible dye terminator,paired-end sequencing, near-term sequencing, exonuclease sequencing,sequencing by ligation, short-read sequencing, single-moleculesequencing, sequencing-by-synthesis, real-time sequencing,reverse-terminator sequencing, nanopore sequencing, 454 sequencing,Solexa Genome Analyzer sequencing, SOLiD™ sequencing, MS-PET sequencing,and any combinations thereof.

Sequence analysis of the nucleic acid molecules (including barcodednucleic acid molecules or derivatives thereof) can be direct orindirect. Thus, the sequence analysis substrate (which can be viewed asthe molecule which is subjected to the sequence analysis step orprocess) can directly be the barcoded nucleic acid molecule or it can bea molecule which is derived therefrom (e.g., a complement thereof).Thus, for example, in the sequence analysis step of a sequencingreaction, the sequencing template can be the barcoded nucleic acidmolecule or it can be a molecule derived therefrom. For example, a firstand/or second strand DNA molecule can be directly subjected to sequenceanalysis (e.g., sequencing), i.e., can directly take part in thesequence analysis reaction or process (e.g., the sequencing reaction orsequencing process, or be the molecule which is sequenced or otherwiseidentified). Alternatively, the barcoded nucleic acid molecule can besubjected to a step of second strand synthesis or amplification beforesequence analysis (e.g., sequencing or identification by anothertechnique). The sequence analysis substrate (e.g., template) can thus bean amplicon or a second strand of a barcoded nucleic acid molecule.

In some embodiments, both strands of a double stranded molecule can besubjected to sequence analysis (e.g., sequenced). In some embodiments,single stranded molecules (e.g., barcoded nucleic acid molecules) can beanalyzed (e.g., sequenced). To perform single molecule sequencing, thenucleic acid strand can be modified at the 3′ end.

In some embodiments, massively parallel pyrosequencing techniques can beused for sequencing nucleic acids. In pyrosequencing, the nucleic acidis amplified inside water droplets in an oil solution (emulsion PCR),with each droplet containing a single nucleic acid template attached toa single primer-coated bead that then forms a clonal colony. Thesequencing system contains many picolitre-volume wells each containing asingle bead and sequencing enzymes. Pyrosequencing uses luciferase togenerate light for detection of the individual nucleotides added to thenascent nucleic acid and the combined data are used to generate sequencereads.

As another example application of pyrosequencing, released PPi can bedetected by being immediately converted to adenosine triphosphate (ATP)by ATP sulfurylase, and the level of ATP generated can be detected vialuciferase-produced photons, such as described in Ronaghi, et al., Anal.Biochem. 242(1), 84-9 (1996); Ronaghi, Genome Res. 11(1), 3-11 (2001);Ronaghi et al. Science 281 (5375), 363 (1998); and U.S. Pat. Nos.6,210,891, 6,258,568, and 6,274,320, the entire contents of each ofwhich are incorporated herein by reference.

Massively parallel sequencing techniques can be used for sequencingnucleic acids, as described above. In one embodiment, a massivelyparallel sequencing technique can be based on reversibledye-terminators. As an example, DNA molecules are first attached toprimers on, e.g., a glass or silicon substrate, and amplified so thatlocal clonal colonies are formed (e.g., by bridge amplification). Fourtypes of ddNTPs are added, and non-incorporated nucleotides are washedaway. Unlike pyrosequencing, the DNA is only extended one nucleotide ata time due to a blocking group (e.g., 3′ blocking group present on thesugar moiety of the ddNTP). A detector acquires images of thefluorescently labelled nucleotides, and then the dye along with theterminal 3′ blocking group is chemically removed from the DNA, as aprecursor to a subsequent cycle. This process can be repeated until therequired sequence data is obtained.

In some embodiments, sequencing is performed by detection of hydrogenions that are released during the polymerization of DNA. A microwellcontaining a template DNA strand to be sequenced can be flooded with asingle type of nucleotide. If the introduced nucleotide is complementaryto the leading template nucleotide, it is incorporated into the growingcomplementary strand. This causes the release of a hydrogen ion thattriggers a hypersensitive ion sensor, which indicates that a reactionhas occurred. If homopolymer repeats are present in the templatesequence, multiple nucleotides will be incorporated in a single cycle.This leads to a corresponding number of released hydrogen ions and aproportionally higher electronic signal.

In some embodiments, sequencing can be performed in situ. In situsequencing methods are particularly useful, for example, when thebiological sample remains intact after analytes on the sample surface(e.g., cell surface analytes) or within the sample (e.g., intracellularanalytes) have been barcoded. In situ sequencing typically involvesincorporation of a labeled nucleotide (e.g., fluorescently labeledmononucleotides or dinucleotides) in a sequential, template-dependentmanner or hybridization of a labeled primer (e.g., a labeled randomhexamer) to a nucleic acid template such that the identities (i.e.,nucleotide sequence) of the incorporated nucleotides or labeled primerextension products can be determined, and consequently, the nucleotidesequence of the corresponding template nucleic acid. Aspects of in situsequencing are described, for example, in Mitra et al., (2003) Anal.Biochem. 320, 55-65, and Lee et al., (2014) Science, 343(6177),1360-1363, the entire contents of each of which are incorporated hereinby reference.

In addition, examples of methods and systems for performing in situsequencing are described in PCT Pat. Application Publication Nos.WO2014/163886, WO2018/045181, WO2018/045186, and in U.S. Pat. Nos.10,138,509 and 10,179,932, the entire contents of each of which areincorporated herein by reference. Example techniques for in situsequencing include, but are not limited to, STARmap (described forexample in Wang et al., (2018) Science, 361(6499) 5691), MERFISH(described for example in Moffitt, (2016) Methods in Enzymology, 572,1-49), and FISSEQ (described for example in U.S. Patent ApplicationPublication No. 2019/0032121). The entire contents of each of theforegoing references are incorporated herein by reference.

For analytes that have been barcoded via partitioning, barcoded nucleicacid molecules or derivatives thereof (e.g., barcoded nucleic acidmolecules to which one or more functional sequences have been added, orfrom which one or more features have been removed) can be pooled andprocessed together for subsequent analysis such as sequencing on highthroughput sequencers. Processing with pooling can be implemented usingbarcode sequences. For example, barcoded nucleic acid molecules of agiven partition can have the same barcode, which is different frombarcodes of other spatial partitions. Alternatively, barcoded nucleicacid molecules of different partitions can be processed separately forsubsequent analysis (e.g., sequencing).

In some embodiments, where capture probes do not contain a spatialbarcode, the spatial barcode can be added after the capture probecaptures analytes from a biological sample and before analysis of theanalytes. When a spatial barcode is added after an analyte is captured,the barcode can be added after amplification of the analyte (e.g.,reverse transcription and polymerase amplification of RNA). In someembodiments, analyte analysis uses direct sequencing of one or morecaptured analytes, such as direct sequencing of hybridized RNA. In someembodiments, direct sequencing is performed after reverse transcriptionof hybridized RNA. In some embodiments direct sequencing is performedafter amplification of reverse transcription of hybridized RNA.

In some embodiments, direct sequencing of captured RNA is performed bysequencing-by-synthesis (SBS). In some embodiments, a sequencing primeris complementary to a sequence in one or more of the domains of acapture probe (e.g., functional domain). In such embodiments,sequencing-by-synthesis can include reverse transcription and/oramplification in order to generate a template sequence (e.g., functionaldomain) from which a primer sequence can bind.

SBS can involve hybridizing an appropriate primer, sometimes referred toas a sequencing primer, with the nucleic acid template to be sequenced,extending the primer, and detecting the nucleotides used to extend theprimer. Preferably, the nucleic acid used to extend the primer isdetected before a further nucleotide is added to the growing nucleicacid chain, thus allowing base-by-base in situ nucleic acid sequencing.The detection of incorporated nucleotides is facilitated by includingone or more labelled nucleotides in the primer extension reaction. Toallow the hybridization of an appropriate sequencing primer to thenucleic acid template to be sequenced, the nucleic acid template shouldnormally be in a single stranded form. If the nucleic acid templatesmaking up the nucleic acid features are present in a double strandedform these can be processed to provide single stranded nucleic acidtemplates using methods well known in the art, for example bydenaturation, cleavage, etc. The sequencing primers which are hybridizedto the nucleic acid template and used for primer extension arepreferably short oligonucleotides, for example, 15 to 25 nucleotides inlength. The sequencing primers can be greater than 25 nucleotides inlength as well. For example, sequencing primers can be about 20 to about60 nucleotides in length, or more than 60 nucleotides in length. Thesequencing primers can be provided in solution or in an immobilizedform. Once the sequencing primer has been annealed to the nucleic acidtemplate to be sequenced by subjecting the nucleic acid template andsequencing primer to appropriate conditions, primer extension is carriedout, for example using a nucleic acid polymerase and a supply ofnucleotides, at least some of which are provided in a labelled form, andconditions suitable for primer extension if a suitable nucleotide isprovided.

Preferably after each primer extension step, a washing step is includedin order to remove unincorporated nucleotides which can interfere withsubsequent steps. Once the primer extension step has been carried out,the nucleic acid colony is monitored to determine whether a labellednucleotide has been incorporated into an extended primer. The primerextension step can then be repeated to determine the next and subsequentnucleotides incorporated into an extended primer. If the sequence beingdetermined is unknown, the nucleotides applied to a given colony areusually applied in a chosen order which is then repeated throughout theanalysis, for example dATP, dTTP, dCTP, dGTP.

SBS techniques which can be used are described for example, but notlimited to, those in U.S. Pat. App. Pub. No. 2007/0166705, U.S. Pat.App. Pub. No. 2006/0188901, U.S. Pat. 7,057,026, U.S. Pat. App. Pub. No.2006/0240439, U.S. Pat. App. Pub. No. 2006/0281109, PCT Pat. App. Pub.No. WO 05/065814, U.S. Pat. App. Pub. No. 2005/0100900, PCT Pat. App.Pub. No. WO 06/064199, PCT Pat. App. Pub. No. WO07/010,251, U.S. Pat.App. Pub. No. 2012/0270305, U.S. Pat. App. Pub. No. 2013/0260372, andU.S. Pat. App. Pub. No. 2013/0079232, the entire contents of each ofwhich are incorporated herein by reference.

In some embodiments, direct sequencing of captured RNA is performed bysequential fluorescence hybridization (e.g., sequencing byhybridization). In some embodiments, a hybridization reaction where RNAis hybridized to a capture probe is performed in situ. In someembodiments, captured RNA is not amplified prior to hybridization with asequencing probe. In some embodiments, RNA is amplified prior tohybridization with sequencing probes (e.g., reverse transcription tocDNA and amplification of cDNA). In some embodiments, amplification isperformed using single-molecule hybridization chain reaction. In someembodiments, amplification is performed using rolling chainamplification.

Sequential fluorescence hybridization can involve sequentialhybridization of probes including degenerate primer sequences and adetectable label. A degenerate primer sequence is a shortoligonucleotide sequence which is capable of hybridizing to any nucleicacid fragment independent of the sequence of said nucleic acid fragment.For example, such a method could include the steps of: (a) providing amixture including four probes, each of which includes either A, C, G, orT at the 5′-terminus, further including degenerate nucleotide sequenceof 5 to 11 nucleotides in length, and further including a functionaldomain (e.g., fluorescent molecule) that is distinct for probes with A,C, G, or T at the 5′-terminus; (b) associating the probes of step (a) tothe target polynucleotide sequences, whose sequence needs will bedetermined by this method; (c) measuring the activities of the fourfunctional domains and recording the relative spatial location of theactivities; (d) removing the reagents from steps (a)-(b) from the targetpolynucleotide sequences; and repeating steps (a)-(d) for n cycles,until the nucleotide sequence of the spatial domain for each bead isdetermined, with modification that the oligonucleotides used in step (a)are complementary to part of the target polynucleotide sequences and thepositions 1 through n flanking the part of the sequences. Because thebarcode sequences are different, in some embodiments, these additionalflanking sequences are degenerate sequences. The fluorescent signal fromeach spot on the array for cycles 1 through n can be used to determinethe sequence of the target polynucleotide sequences.

In some embodiments, direct sequencing of captured RNA using sequentialfluorescence hybridization is performed in vitro. In some embodiments,captured RNA is amplified prior to hybridization with a sequencing probe(e.g., reverse transcription to cDNA and amplification of cDNA). In someembodiments, a capture probe containing captured RNA is exposed to thesequencing probe targeting coding regions of RNA. In some embodiments,one or more sequencing probes are targeted to each coding region. Insome embodiments, the sequencing probe is designed to hybridize withsequencing reagents (e.g., a dye-labeled readout oligonucleotides). Asequencing probe can then hybridize with sequencing reagents. In someembodiments, output from the sequencing reaction is imaged. In someembodiments, a specific sequence of cDNA is resolved from an image of asequencing reaction. In some embodiments, reverse transcription ofcaptured RNA is performed prior to hybridization to the sequencingprobe. In some embodiments, the sequencing probe is designed to targetcomplementary sequences of the coding regions of RNA (e.g., targetingcDNA).

In some embodiments, a captured RNA is directly sequenced using ananopore-based method. In some embodiments, direct sequencing isperformed using nanopore direct RNA sequencing in which captured RNA istranslocated through a nanopore. A nanopore current can be recorded andconverted into a base sequence. In some embodiments, captured RNAremains attached to a substrate during nanopore sequencing. In someembodiments, captured RNA is released from the substrate prior tonanopore sequencing. In some embodiments, where the analyte of interestis a protein, direct sequencing of the protein can be performed usingnanopore-based methods. Examples of nanopore-based sequencing methodsthat can be used are described in Deamer et al., Trends Biotechnol. 18,14 7-151 (2000); Deamer et al., Acc. Chem. Res. 35:817-825 (2002); Li etal., Nat. Mater. 2:611-615 (2003); Soni et al., Clin. Chem. 53,1996-2001 (2007); Healy et al., Nanomed. 2, 459-481 (2007); Cockroft etal., J. Am. Chem. Soc. 130, 818-820 (2008); and in U.S. Pat. 7,001,792.The entire contents of each of the foregoing references are incorporatedherein by reference.

In some embodiments, direct sequencing of captured RNA is performedusing single molecule sequencing by ligation. Such techniques utilizeDNA ligase to incorporate oligonucleotides and identify theincorporation of such oligonucleotides. The oligonucleotides typicallyhave different labels that are correlated with the identity of aparticular nucleotide in a sequence to which the oligonucleotideshybridize. Aspects and features involved in sequencing by ligation aredescribed, for example, in Shendure et al. Science (2005), 309:1728-1732, and in U.S. Pat. Nos. 5,599,675; 5,750,341; 6,969,488;6,172,218; and 6,306,597, the entire contents of each of which areincorporated herein by reference.

In some embodiments, nucleic acid hybridization can be used forsequencing. These methods utilize labeled nucleic acid decoder probesthat are complementary to at least a portion of a barcode sequence.Multiplex decoding can be performed with pools of many different probeswith distinguishable labels. Non-limiting examples of nucleic acidhybridization sequencing are described for example in U.S. Pat. No.8,460,865, and in Gunderson et al., Genome Research 14:870-877 (2004),the entire contents of each of which are incorporated herein byreference.

In some embodiments, commercial high-throughput digital sequencingtechniques can be used to analyze barcode sequences, in which DNAtemplates are prepared for sequencing not one at a time, but in a bulkprocess, and where many sequences are read out preferably in parallel,or alternatively using an ultra-high throughput serial process thatitself may be parallelized. Examples of such techniques includeIllumina^(®) sequencing (e.g., flow cell-based sequencing techniques),sequencing by synthesis using modified nucleotides (such ascommercialized in TruSeq™ and HiSeq™ technology by Illumina, Inc., SanDiego, CA), HeliScope™ by Helicos Biosciences Corporation, Cambridge,MA, and PacBio RS by Pacific Biosciences of California, Inc., MenloPark, CA), sequencing by ion detection technologies (Ion Torrent, Inc.,South San Francisco, CA), and sequencing of DNA nanoballs (CompleteGenomics, Inc., Mountain View, CA).

In some embodiments, detection of a proton released upon incorporationof a nucleotide into an extension product can be used in the methodsdescribed herein. For example, the sequencing methods and systemsdescribed in U.S. Pat. Application Publication Nos. 2009/0026082,2009/0127589, 2010/0137143, and 2010/0282617, can be used to directlysequence barcodes. The entire contents of each of the foregoingreferences are incorporated herein by reference.

In some embodiments, real-time monitoring of DNA polymerase activity canbe used during sequencing. For example, nucleotide incorporations can bedetected through fluorescence resonance energy transfer (FRET), asdescribed for example in Levene et al., Science (2003), 299, 682-686,Lundquist et al., Opt. Lett. (2008), 33, 1026-1028, and Korlach et al.,Proc. Natl. Acad. Sci. USA (2008), 105, 1176-1181. The entire contentsof each of the foregoing references are herein incorporated byreference.

IV. Multiplexing (A) Multiplexing Generally

In various embodiments of spatial analysis as described herein, featurescan include different types of capture probes for analyzing bothintrinsic and extrinsic information for individual cells. For example, afeature can include one or more of the following: 1) a capture probefeaturing a capture domain that binds to one or more endogenous nucleicacids in the cell; 2) a capture probe featuring a capture domain thatbinds to one or more exogenous nucleic acids in the cell (e.g., nucleicacids from a microorganism (e.g., a virus, a bacterium)) that infectsthe cell, nucleic acids introduced into the cell (e.g., such as plasmidsor nucleic acid derived therefrom), nucleic acids for gene editing(e.g., CRISPR-related RNA such as crRNA, guide RNA); 3) a capture probefeaturing a capture domain that binds to an analyte capture agent (e.g.,an antibody coupled to a oligonucleotide that includes a capture agentbarcode domain having an analyte capture sequence that binds the capturedomain), and 4) a capture moiety featuring a domain that binds to aprotein (e.g., an exogenous protein expressed in the cell, a proteinfrom a microorganism (e.g., a virus, a bacterium)) that infects thecell, or a binding partner for a protein of the cell (e.g., an antigenfor an immune cell receptor).

In some embodiments of any of the spatial analysis methods as describedherein, spatial profiling includes concurrent analysis of two differenttypes of analytes. A feature can be a gel bead, which is coupled (e.g.,reversibly coupled) to one or more capture probes. The capture probescan include a spatial barcode sequence and a poly(T) priming sequencethat can hybridize with the poly(A) tail of an mRNA transcript. Thecapture probe can also include a UMI sequence that can uniquely identifya given transcript. The capture probe can also include a spatial barcodesequence and a random N-mer priming sequence that is capable of randomlyhybridizing with gDNA. In this configuration, capture probes can includethe same spatial barcode sequence, which permits association ofdownstream sequencing reads with the feature.

In some embodiments of any of the spatial analysis methods as describedherein, a feature can be a gel bead, which is coupled (e.g., reversiblycoupled) to capture probes. The Capture probe can include a spatialbarcode sequence and a poly(T) priming sequence that can hybridize withthe poly(A) tail of an mRNA transcript. The capture probe can alsoinclude a UMI sequence that can uniquely identify a given transcript.The capture probe can include a spatial barcode sequence and a capturedomain that is capable of specifically hybridizing with an analytecapture agent. The analyte capture agent can includes an oligonucleotidethat includes an analyte capture sequence that interacts with thecapture domain coupled to the feature. The oligonucleotide of theanalyte capture agent can be coupled to an antibody that is bound to thesurface of a cell. The oligonucleotide includes a barcode sequence(e.g., an analyte binding moiety barcode) that uniquely identifies theantibody (and thus, the particular cell surface feature to which it isbound). In this configuration, the capture probes include the samespatial barcode sequence, which permit downstream association ofbarcoded nucleic acids with the location on the spatial array. In someembodiments of any of the spatial profiling methods described herein,the analyte capture agents can be can be produced by any suitable route,including via example coupling schemes described elsewhere herein.

In some embodiments of any of the spatial analysis methods describedherein, other combinations of two or more biological analytes that canbe concurrently measured include, without limitation: (a) genomic DNAand cell surface features (e.g., via analyte capture agents that bind toa cell surface feature), (b) mRNA and a lineage tracing construct, (c)mRNA and cell methylation status, (d) mRNA and accessible chromatin(e.g., ATAC-seq, DNase-seq, and/or MNase-seq), (e) mRNA and cell surfaceor intracellular proteins and/or metabolites, (f) mRNA and chromatin(spatial organization of chromatin in a cell), (g) an analyte captureagent (e.g., any of the MHC multimers described herein) and a V(D)Jsequence of an immune cell receptor (e.g., T-cell receptor), (h) mRNAand a perturbation agent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc fingernuclease, and/or antisense oligonucleotide as described herein), (i)genomic DNA and a perturbation agent, (j) an analyte capture agent and aperturbation agents, (k) accessible chromatin and a perturbation agent,(l) chromatin (e.g., spatial organization of chromatin in a cell) and aperturbation agent, and (m) cell surface or intracellular proteinsand/or metabolites and a perturbation agent (e.g., any of theperturbation agents described herein), or any combination thereof.

In some embodiments of any of the spatial analysis methods describedherein, the first analyte can include a nucleic acid molecule with anucleic acid sequence (e.g., mRNA, complementary DNA derived fromreverse transcription of mRNA) encoding at least a portion of a V(D)Jsequence of an immune cell receptor (e.g., a TCR or BCR). In someembodiments, the nucleic acid molecule with a nucleic acid sequenceencoding at least a portion of a V(D)J sequence of an immune cellreceptor is cDNA first generated from reverse transcription of thecorresponding mRNA, using a poly(T) containing primer. The cDNA that isgenerated can then be barcoded using a primer, featuring a spatialbarcode sequence (and optionally, a UMI sequence) that hybridizes withat least a portion of the cDNA that is generated. In some embodiments, atemplate switching oligonucleotide in conjunction a terminal transferaseor a reverse transcriptase having terminal transferase activity can beemployed to generate a priming region on the cDNA to which a barcodedprimer can hybridize during cDNA generation. Terminal transferaseactivity can, for example, add a poly(C) tail to a 3′ end of the cDNAsuch that the template switching oligonucleotide can bind via a poly(G)priming sequence and the 3′ end of the cDNA can be further extended. Theoriginal mRNA template and template switching oligonucleotide can thenbe denatured from the cDNA and the barcoded primer comprising a sequencecomplementary to at least a portion of the generated priming region onthe cDNA can then hybridize with the cDNA and a barcoded constructcomprising the barcode sequence (and any optional UMI sequence) and acomplement of the cDNA generated. Additional methods and compositionssuitable for barcoding cDNA generated from mRNA transcripts includingthose encoding V(D)J regions of an immune cell receptor and/or barcodingmethods and composition including a template switch oligonucleotide aredescribed, for example, in PCT Pat. Application Publication No. WO2018/075693, and in U.S. Pat. Application Publication No. 2018/0105808,the entire contents of each of which are incorporated herein byreference.

In some embodiments, V(D)J analysis can be performed using methodssimilar to those described herein. For example, V(D)J analysis can becompleted with the use of one or more analyte capture agents that bindto particular surface features of immune cells and are associated withbarcode sequences (e.g., analyte binding moiety barcodes). The one ormore analyte capture agents can include an MHC or MHC multimer. Abarcoded oligonucleotide coupled to a bead that can be used for V(D)Janalysis. The oligonucleotide is coupled to a bead by a releasablelinkage, such as a disulfide linker. The oligonucleotide can includefunctional sequences that are useful for subsequent processing, such asfunctional sequence, which can include a sequencer specific flow cellattachment sequence, e.g., a P5 sequence, as well as functionalsequence, which can include sequencing primer sequences, e.g., a R1primer binding site. In some embodiments, the sequence can include a P7sequence and a R2 primer binding site. A barcode sequence can beincluded within the structure for use in barcoding the templatepolynucleotide. The functional sequences can be selected forcompatibility with a variety of different sequencing systems, e.g., 454Sequencing, Ion Torrent Proton or PGM, Illumina X10, etc., and therequirements thereof. In some embodiments, the barcode sequence,functional sequences (e.g., flow cell attachment sequence) andadditional sequences (e.g., sequencing primer sequences) can be commonto all of the oligonucleotides attached to a given bead. The barcodedoligonucleotide can also include a sequence to facilitate templateswitching (e.g., a poly(G) sequence). In some embodiments, theadditional sequence provides a unique molecular identifier (UMI)sequence segment, as described elsewhere herein.

In an exemplary method of cellular polynucleotide analysis using abarcode oligonucleotide, a cell is co-partitioned along with a beadbearing a barcoded oligonucleotide and additional reagents such as areverse transcriptase, primers, oligonucleotides (e.g., templateswitching oligonucleotides), dNTPs, and a reducing agent into apartition (e.g., a droplet in an emulsion). Within the partition, thecell can be lysed to yield a plurality of template polynucleotides(e.g., DNA such as genomic DNA, RNA such as mRNA, etc.).

A reaction mixture featuring a template polynucleotide from a cell and(i) the primer having a sequence towards a 3′ end that hybridizes to thetemplate polynucleotide (e.g., poly(T)) and (ii) a template switchingoligonucleotide that includes a first oligonucleotide towards a 5′ endcan be subjected to an amplification reaction to yield a firstamplification product. In some embodiments, the template polynucleotideis an mRNA with a poly(A) tail and the primer that hybridizes to thetemplate polynucleotide includes a poly(T) sequence towards a 3′ end,which is complementary to the poly(A) segment. The first oligonucleotidecan include at least one of an adaptor sequence, a barcode sequence, aunique molecular identifier (UMI) sequence, a primer binding site, and asequencing primer binding site or any combination thereof. In somecases, a first oligonucleotide is a sequence that can be common to allpartitions of a plurality of partitions. For example, the firstoligonucleotide can include a flow cell attachment sequence, anamplification primer binding site, or a sequencing primer binding siteand the first amplification reaction facilitates the attachment theoligonucleotide to the template polynucleotide from the cell. In someembodiments, the first oligonucleotide includes a primer binding site.In some embodiments, the first oligonucleotide includes a sequencingprimer binding site.

The sequence towards a 3′ end (e.g., poly(T)) of the primer hybridizesto the template polynucleotide. In a first amplification reaction,extension reaction reagents, e.g., reverse transcriptase, nucleosidetriphosphates, co-factors (e.g., Mg²⁺ or Mn²⁺), that are alsoco-partitioned, can extend the primer sequence using the cell’s nucleicacid as a template, to produce a transcript, e.g., cDNA, having afragment complementary to the strand of the cell’s nucleic acid to whichthe primer annealed. In some embodiments, the reverse transcriptase hasterminal transferase activity and the reverse transcriptase addsadditional nucleotides, e.g., poly(C), to the cDNA in a templateindependent manner.

The template switching oligonucleotide, for example a template switchingoligonucleotide which includes a poly(G) sequence, can hybridize to thecDNA and facilitate template switching in the first amplificationreaction. The transcript, therefore, can include the sequence of theprimer, a sequence complementary to the template polynucleotide from thecell, and a sequence complementary to the template switchingoligonucleotide.

In some embodiments of any of the spatial analysis methods describedherein, subsequent to the first amplification reaction, the firstamplification product or transcript can be subjected to a secondamplification reaction to generate a second amplification product. Insome embodiments, additional sequences (e.g., functional sequences suchas flow cell attachment sequence, sequencing primer binding sequences,barcode sequences, etc.) are attached. The first and secondamplification reactions can be performed in the same volume, such as forexample in a droplet. In some embodiments, the first amplificationproduct is subjected to a second amplification reaction in the presenceof a barcoded oligonucleotide to generate a second amplification producthaving a barcode sequence. The barcode sequence can be unique to apartition, that is, each partition can have a unique barcode sequence.The barcoded oligonucleotide can include a sequence of at least asegment of the template switching oligonucleotide and at least a secondoligonucleotide. The segment of the template switching oligonucleotideon the barcoded oligonucleotide can facilitate hybridization of thebarcoded oligonucleotide to the transcript, e.g., cDNA, to facilitatethe generation of a second amplification product. In addition to abarcode sequence, the barcoded oligonucleotide can include a secondoligonucleotide such as at least one of an adaptor sequence, a uniquemolecular identifier (UMI) sequence, a primer binding site, and asequencing primer binding site, or any combination thereof.

In some embodiments of any of the spatial analysis methods describedherein, the second amplification reaction uses the first amplificationproduct as a template and the barcoded oligonucleotide as a primer. Insome embodiments, the segment of the template switching oligonucleotideon the barcoded oligonucleotide can hybridize to the portion of the cDNAor complementary fragment having a sequence complementary to thetemplate switching oligonucleotide or that which was copied from thetemplate switching oligonucleotide. In the second amplificationreaction, extension reaction reagents, e.g., polymerase, nucleosidetriphosphates, co-factors (e.g., Mg²⁺ or Mn²⁺), that are alsoco-partitioned, can extend the primer sequence using the firstamplification product as template. The second amplification product caninclude a second oligonucleotide, a sequence of a segment of thetemplate polynucleotide (e.g., mRNA), and a sequence complementary tothe primer.

In some embodiments of any of the spatial analysis methods describedherein, the second amplification product uses the barcodedoligonucleotide as a template and at least a portion of the firstamplification product as a primer. The segment of the firstamplification product (e.g., cDNA) having a sequence complementary tothe template switching oligonucleotide can hybridize to the segment ofthe barcoded oligonucleotide comprising a sequence of at least a segmentof the template switching oligonucleotide. In the second amplificationreaction, extension reaction reagents, e.g., polymerase, nucleosidetriphosphates, co-factors (e.g., Mg²⁺ or Mn²⁺), that are alsoco-partitioned, can extend the primer sequence (e.g., firstamplification product) using the barcoded oligonucleotide as template.The second amplification product can include the sequence of the primer,a sequence which is complementary to the sequence of the templatepolynucleotide (e.g., mRNA), and a sequence complementary to the secondoligonucleotide.

In some embodiments of any of the spatial analysis methods describedherein, three or more classes of biological analytes can be concurrentlymeasured. For example, a feature can include capture probes that canparticipate in an assay of at least three different types of analytesvia three different capture domains. A bead can be coupled to a barcodedoligonucleotide that includes a capture domain that includes a poly(T)priming sequence for mRNA analysis; a barcoded oligonucleotide thatincludes a capture domain that includes a random N-mer priming sequencefor gDNA analysis; and a barcoded oligonucleotide that includes acapture domain that can specifically bind a an analyte capture agent(e.g., an antibody with a spatial barcode), via its analyte capturesequence.

In some embodiments of any of the spatial analysis methods describedherein, other combinations of three or more biological analytes that canbe concurrently measured include, without limitation: (a) mRNA, alineage tracing construct, and cell surface and/or intracellularproteins and/or metabolites; (b) mRNA, accessible chromatin (e.g.,ATAC-seq, DNase-seq, and/or MNase-seq), and cell surface and/orintracellular proteins and/or metabolites; (c) mRNA, genomic DNA, and aperturbation reagent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc fingernuclease, and/or antisense oligonucleotide as described herein); (d)mRNA, accessible chromatin, and a perturbation reagent; (e) mRNA, ananalyte capture agent (e.g., any of the MHC multimers described herein),and a perturbation reagent; (f) mRNA, cell surface and/or intracellularproteins and/or metabolites, and a perturbation agent; (g) mRNA, a V(D)Jsequence of an immune cell receptor (e.g., T-cell receptor), and aperturbation reagent; (h) mRNA, an analyte capture agent, and a V(D)Jsequence of an immune cell receptor; (i) cell surface and/orintracellular proteins and/or metabolites, a an analyte capture agent(e.g., the MHC multimers described herein), and a V(D)J sequence of animmune cell receptor; (j) methylation status, mRNA, and cell surfaceand/or intracellular proteins and/or metabolites; (k) mRNA, chromatin(e.g., spatial organization of chromatin in a cell), and a perturbationreagent; (l) a V(D)J sequence of an immune cell receptor, chromatin(e.g., spatial organization of chromatin in a cell); and a perturbationreagent; and (m) mRNA, a V(D)J sequence of an immune cell receptor, andchromatin (e.g., spatial organization of chromatin in a cell), or anycombination thereof.

In some embodiments of any of the spatial analysis methods describedherein, four or more classes biological analytes can be concurrentlymeasured. A feature can be a bead that is coupled to barcoded primersthat can each participate in an assay of a different type of analyte.The feature is coupled (e.g., reversibly coupled) to a capture probethat includes a capture domain that includes a poly(T) priming sequencefor mRNA analysis and is also coupled (e.g., reversibly coupled) tocapture probe that includes a capture domain that includes a randomN-mer priming sequence for gDNA analysis. Moreover, the feature is alsocoupled (e.g., reversibly coupled) to a capture probe that binds ananalyte capture sequence of an analyte capture agent via its capturedomain. The feature can also be coupled (e.g., reversibly coupled) to acapture probe that can specifically bind a nucleic acid molecule thatcan function as a perturbation agent (e.g., a CRISPR crRNA/sgRNA, TALEN,zinc finger nuclease, and/or antisense oligonucleotide as describedherein), via its capture domain.

In some embodiments of any of the spatial analysis methods describedherein, each of the various spatially-barcoded capture probes present ata given feature or on a given bead include the same spatial barcodesequence. In some embodiments, each barcoded capture probe can bereleased from the feature in a manner suitable for analysis of itsrespective analyte. For example, barcoded constructs A, B, C and D canbe generated as described elsewhere herein and analyzed. Barcodedconstruct A can include a sequence corresponding to the barcode sequencefrom the bead (e.g., a spatial barcode) and a DNA sequence correspondingto a target mRNA. Barcoded construct B can include a sequencecorresponding to the barcode sequence from the bead (e.g., a spatialbarcode) and a sequence corresponding to genomic DNA. Barcoded constructC can include a sequence corresponding to the barcode sequence from thebead (e.g., a spatial barcode) and a sequence corresponding to barcodesequence associated with an analyte capture agent (e.g., an analytebinding moiety barcode). Barcoded construct D can include a sequencecorresponding to the barcode sequence from the bead (e.g., a spatialbarcode) and a sequence corresponding to a CRISPR nucleic acid (which,in some embodiments, also includes a barcode sequence). Each constructcan be analyzed (e.g., via any of a variety of sequencing methods) andthe results can be associated with the given cell from which the variousanalytes originated. Barcoded (or even non-barcoded) constructs can betailored for analyses of any given analyte associated with a nucleicacid and capable of binding with such a construct.

In some embodiments of any of the spatial analysis methods describedherein, other combinations of four or more biological analytes that canbe concurrently measured include, without limitation: (a) mRNA, alineage tracing construct, cell surface and/or intracellular proteinsand/or metabolites, and gDNA; (b) mRNA, accessible chromatin (e.g.,ATAC-seq, DNase-seq, and/or MNase-seq), cell surface and/orintracellular proteins and/or metabolites, and a perturbation agent(e.g., a CRISPR crRNA/sgRNA, TALEN, zinc finger nuclease, and/orantisense oligonucleotide as described herein); (c) mRNA, cell surfaceand/or intracellular proteins and/or metabolites, an analyte captureagent (e.g., the MHC multimers described herein), and a V(D)J sequenceof an immune cell receptor (e.g., T-cell receptor); (d) mRNA, genomicDNA, a perturbation reagent, and accessible chromatin; (e) mRNA, cellsurface and/or intracellular proteins and/or metabolites, an analytecapture agent (e.g., the MHC multimers described herein), and aperturbation reagent; (f) mRNA, cell surface and/or intracellularproteins and/or metabolites, a perturbation reagent, and a V(D)Jsequence of an immune cell receptor (e.g., T-cell receptor); (g) mRNA, aperturbation reagent, an analyte capture agent (e.g., the MHC multimersdescribed herein), and a V(D)J sequence of an immune cell receptor(e.g., T-cell receptor); (h) mRNA, chromatin (e.g., spatial organizationof chromatin in a cell), and a perturbation reagent; (i) a V(D)Jsequence of an immune cell receptor, chromatin (e.g., spatialorganization of chromatin in a cell); and a perturbation reagent; (j)mRNA, a V(D)J sequence of an immune cell receptor, chromatin (e.g.,spatial organization of chromatin in a cell), and genomic DNA; (k) mRNA,a V(D)J sequence of an immune cell receptor, chromatin (e.g., spatialorganization of chromatin in a cell), and a perturbation reagent, or anycombination thereof.

(B) Construction of Spatial Arrays for Multi-Analyte Analysis

This disclosure also provides methods and materials for constructing aspatial array capable of multi-analyte analysis. In some embodiments, aspatial array includes a plurality of features on a substrate where oneor more members of the plurality of features include a plurality ofoligonucleotides having a first type functional sequence andoligonucleotides having a second, different type of functional sequence.In some embodiments, a feature can include oligonucleotides with twotypes of functional sequences. A feature can be coupled tooligonucleotides comprising a TruSeq functional sequence and also tooligonucleotides comprising a Nextera functional sequence. In someembodiments, one or more members of the plurality of features comprisesboth types of functional sequences. In some embodiments, one or moremembers of the plurality features includes a first type of functionalsequence. In some embodiments, one or more members of the plurality offeatures includes a second type of functional sequence. In someembodiments, an additional oligonucleotide can be added to thefunctional sequence to generate a full oligonucleotide where the fulloligonucleotide includes a spatial barcode sequence, an optional UMIsequence, a priming sequence, and a capture domain. Attachment of thesesequences can be via ligation (including via splint ligation as isdescribed in U.S. Patent Application Publication No. 20140378345, theentire contents of which are incorporated herein by reference), or anyother suitable route. As discussed herein, oligonucleotides can behybridized with splint sequences that can be helpful in constructingcomplete full oligonucleotides (e.g., oligonucleotides that are capableof spatial analysis).

In some embodiments, the oligonucleotides that hybridize to thefunctional sequences (e.g., TruSeq and Nextera) located on the featuresinclude capture domains capable of capturing different types of analytes(e.g., mRNA, genomic DNA, cell surface proteins, or accessiblechromatin). In some examples, oligonucleotides that can bind to theTruSeq functional sequences can include capture domains that includepoly(T) capture sequences. In addition to the poly(T) capture sequences,the oligonucleotides that can bind the TruSeq functional groups can alsoinclude a capture domain that includes a random N-mer sequence forcapturing genomic DNA (e.g., or any other sequence or domain asdescribed herein capable of capturing any of the biological analytesdescribed herein). In such cases, the spatial arrays can be constructedby applying ratios of TruSeq-poly(T) and TruSeq—N—mer oligonucleotidesto the features comprising the functional TruSeq sequences. This canproduce spatial arrays where a portion of the oligonucleotides cancapture mRNA and a different portion of oligonucleotides can capturegenomic DNA. In some embodiments, one or more members of a plurality offeatures include both TruSeq and Nextera functional sequences. In suchcases, a feature including both types of functional sequences is capableof binding oligonucleotides specific to each functional sequence. Forexample, an oligonucleotide capable of binding to a TruSeq functionalsequence could be used to deliver an oligonucleotide including a poly(T)capture domain and an oligonucleotide capable of binding to a Nexterafunctional sequence could be used to deliver an oligonucleotideincluding an N-mer capture domain for capturing genomic DNA. It will beappreciated by a person of ordinary skill in the art that anycombination of capture domains (e.g., capture domains having any of thevariety of capture sequences described herein capable of binding to anyof the different types of analytes as described herein) could becombined with oligonucleotides capable of binding to TruSeq and Nexterafunctional sequences to construct a spatial array.

In some embodiments, an oligonucleotide that includes a capture domain(e.g., an oligonucleotide capable of coupling to an analyte) or ananalyte capture agent can include an oligonucleotide sequence that iscapable of binding or ligating to an assay primer. The adapter can allowthe capture probe or the analyte capture agent to be attached to anysuitable assay primers and used in any suitable assays. The assay primercan include a priming region and a sequence that is capable of bindingor ligating to the adapter. In some embodiments, the adapter can be anon-specific primer (e.g., a 5′ overhang) and the assay primer caninclude a 3′ overhang that can be ligated to the 5′ overhang. Thepriming region on the assay primer can be any primer described herein,e.g., a poly (T) primer, a random N-mer primer, a target-specificprimer, or an analyte capture agent capture sequence.

In some examples, an oligonucleotide can includes an adapter, e.g., a 5′overhang with 10 nucleotides. The adapter can be ligated to assayprimers, each of which includes a 3′ overhang with 10 nucleotides thatcomplementary to the 5′ overhang of the adapter. The capture probe canbe used in any assay by attaching to the assay primer designed for thatassay.

Adapters and assay primers can be used to allow the capture probe or theanalyte capture agent to be attached to any suitable assay primers andused in any suitable assays. A capture probe that includes a spatialbarcode can be attached to a bead that includes a poly(dT) sequence. Acapture probe including a spatial barcode and a poly(T) sequence can beused to assay multiple biological analytes as generally described herein(e.g., the biological analyte includes a poly(A) sequence or is coupledto or otherwise is associated with an analyte capture agent comprising apoly(A) sequence as the analyte capture sequence).

A splint oligonucleotide with a poly(A) sequence can be used tofacilitate coupling to a capture probe that includes a spatial barcodeand a second sequence that facilitates coupling with an assay primer.Assay primers include a sequence complementary to the splint oligosecond sequence and an assay-specific sequence that determines assayprimer functionality (e.g., a poly(T) primer, a random N-mer primer, atarget-specific primer, or an analyte capture agent capture sequence asdescribed herein).

In some embodiments of any of the spatial profiling methods describedherein, a feature can include a capture probe that includes a spatialbarcode comprising a switch oligonucleotide, e.g., with a 3′ end 3rG.For example, a feature (e.g., a gel bead) with a spatial barcodefunctionalized with a 3rG sequence can be used that enables templateswitching (e.g., reverse transcriptase template switching), but is notspecific for any particular assay. In some embodiments, the assayprimers added to the reaction can determine which type of analytes areanalyzed. For example, the assay primers can include binding domainscapable of binding to target biological analytes (e.g., poly (T) formRNA, N-mer for genomic DNA, etc.). A capture probe (e.g., anoligonucleotide capable of spatial profiling) can be generated by usinga reverse transcriptase enzyme/polymerase to extend, which is followedby template switching onto the barcoded adapter oligonucleotide toincorporate the barcode and other functional sequences. In someembodiments, the assay primers include capture domains capable ofbinding to a poly(T) sequence for mRNA analysis, random primers forgenomic DNA analysis, or a capture sequence that can bind a nucleic acidmolecule coupled to an analyte binding moiety (e.g., a an analytecapture sequence of an analyte capture agent) or a nucleic acid moleculethat can function in as a perturbation reagent (e.g., a CRISPRcrRNA/sgRNA, TALEN, zinc finger nuclease, and/or antisenseoligonucleotide as described herein).

V. Systems for Sample Analysis

The methods described above for analyzing biological samples can beimplemented using a variety of hardware components. In this section,examples of such components are described. However, it should beunderstood that in general, the various steps and techniques discussedherein can be performed using a variety of different devices and systemcomponents, not all of which are expressly set forth.

FIG. 22A is a schematic diagram showing an example sample handlingapparatus 2200. Sample handling apparatus 2200 includes a sample chamber2202 that, when closed or sealed, is fluid-tight. Within chamber 2202, afirst holder 2204 holds a first substrate 2206 on which a sample 2208 ispositioned. Sample chamber 2202 also includes a second holder 2210 thatholds a second substrate 2212 with an array of features 2214, asdescribed above.

A fluid reservoir 2216 is connected to the interior volume of samplechamber 2202 via a fluid inlet 2218. Fluid outlet 2220 is also connectedto the interior volume of sample chamber 2202, and to valve 2222. Inturn, valve 2222 is connected to waste reservoir 2224 and, optionally,to analysis apparatus 2226. A control unit 2228 is electricallyconnected to second holder 2210, to valve 2222, to waste reservoir 2224,and to fluid reservoir 2216.

During operation of apparatus 2200, any of the reagents, solutions, andother biochemical components described above can be delivered intosample chamber 2202 from fluid reservoir 2216 via fluid inlet 2218.Control unit 2228, connected to fluid reservoir 2216, can control thedelivery of reagents, solutions, and components, and adjust the volumesand flow rates according to programmed analytical protocols for varioussample types and analysis procedures. In some embodiments, fluidreservoir 2216 includes a pump, which can be controlled by control unit2228, to facilitate delivery of substances into sample chamber 2202.

In certain embodiments, fluid reservoir 2216 includes a plurality ofchambers, each of which is connected to fluid inlet 2218 via a manifold(not shown). Control unit 2228 can selectively deliver substances fromany one or more of the multiple chambers into sample chamber 2202 byadjusting the manifold to ensure that the selected chambers arefluidically connected to fluid inlet 2218.

In general, control unit 2228 can be configured to introduce substancesfrom fluid reservoir 2216 into sample chamber 2202 before, after, orboth before and after, sample 2208 on first substrate 2206 hasinteracted with the array of features 2214 on first substrate 2212. Manyexamples of such substances have been described previously. Examples ofsuch substances include, but are not limited to, permeabilizing agents,buffers, fixatives, staining solutions, washing solutions, and solutionsof various biological reagents (e.g., enzymes, peptides,oligonucleotides, primers).

To initiate interaction between sample 2208 and feature array 2214, thesample and array are brought into spatial proximity. To facilitate thisstep, second holder 2210 - under the control of control unit 2228 - cantranslate second substrate 2212 in any of the x-, y-, and z-coordinatedirections. In particular, control unit 2228 can direct second holder2210 to translate second substrate 2212 in the z-direction so thatsample 2208 contacts, or nearly contacts, feature array 2214.

In some embodiments, apparatus 2200 can optionally include an alignmentsub-system 2230, which can be electrically connected to control unit2228. Alignment sub-system 2230 functions to ensure that sample 2208 andfeature array 2214 are aligned in the x-y plane prior to translatingsecond substrate 2212 in the z-direction so that sample 2208 contacts,or nearly contacts, feature array 2214.

Alignment sub-system 2230 can be implemented in a variety of ways. Insome embodiments, for example, alignment sub-system 2230 includes animaging unit that obtains one or more images showing fiducial markingson first substrate 2206 and/or second substrate 2212. Control unit 2218analyzes the image(s) to determine appropriate translations of secondsubstrate 2212 in the x- and/or y-coordinate directions to ensure thatsample 2208 and feature array 2214 are aligned prior to translation inthe z-coordinate direction.

In certain embodiments, control unit 2228 can optionally regulate theremoval of substances from sample chamber 2202. For example, controlunit 2228 can selectively adjust valve 2222 so that substancesintroduced into sample chamber 2202 from fluid reservoir 2216 aredirected into waste reservoir 2224. In some embodiments, waste reservoir2224 can include a reduced-pressure source (not shown) electricallyconnected to control unit 2228. Control unit 2228 can adjust the fluidpressure in fluid outlet 2220 to control the rate at which fluids areremoved from sample chamber 2202 into waste reservoir 2224.

In some embodiments, analytes from sample 2208 or from feature array2214 can be selectively delivered to analysis apparatus 2226 viasuitable adjustment of valve 2222 by control unit 2228. As describedabove, in some embodiments, analysis apparatus 2226 includes areduced-pressure source (not shown) electrically connected to controlunit 2228, so that control unit 2228 can adjust the rate at whichanalytes are delivered to analysis apparatus 2226. As such, fluid outlet2220 effectively functions as an analyte collector, while analysis ofthe analytes is performed by analysis apparatus 2226. It should be notedthat not all of the workflows and methods described herein areimplemented via analysis apparatus 2226. For example, in someembodiments, analytes that are captured by feature array 2214 remainbound to the array (i.e., are not cleaved from the array), and featurearray 2214 is directly analyzed to identify specifically-bound samplecomponents.

In addition to the components described above, apparatus 2200 canoptionally include other features as well. In some embodiments, forexample, sample chamber 2202 includes a heating sub-system 2232electrically connected to control unit 2228. Control unit 2228 canactivate heating sub-system 2232 to heat sample 2208 and/or featurearray 2214, which can help to facilitate certain steps of the methodsdescribed herein.

In certain embodiments, sample chamber 2202 includes an electrode 2234electrically connected to control unit 2228. Control unit 2228 canoptionally activate electrode 2234, thereby establishing an electricfield between the first and second substrates. Such fields can be used,for example, to facilitate migration of analytes from sample 2208 towardfeature array 2214.

In some of the methods described herein, one or more images of a sampleand/or a feature array are acquired. Imaging apparatus that is used toobtain such images can generally be implemented in a variety of ways.FIG. 22B shows one example of an imaging apparatus 2250. Imagingapparatus 2250 includes a light source 2252, light conditioning optics2254, light delivery optics 2256, light collection optics 2260, lightadjusting optics 2262, and a detection sub-system 2264. Each of theforegoing components can optionally be connected to control unit 2228,or alternatively, to another control unit. For purposes of explanationbelow, it will be assumed that control unit 2228 is connected to thecomponents of imaging apparatus 2250.

During operation of imaging apparatus 2250, light source 2252 generateslight. In general, the light generated by source 2252 can include lightin any one or more of the ultraviolet, visible, and/or infrared regionsof the electromagnetic spectrum. A variety of different light sourceelements can be used to generate the light, including (but not limitedto) light emitting diodes, laser diodes, laser sources, fluorescentsources, incandescent sources, and glow-discharge sources.

The light generated by light source 2252 is received by lightconditioning optics 2254. In general, light conditioning optics 2254modify the light generated by light source 2252 for specific imagingapplications. For example, in some embodiments, light conditioningoptics 2254 modify the spectral properties of the light, e.g., byfiltering out certain wavelengths of the light. For this purpose, lightconditioning optics 2254 can include a variety of spectral opticalelements, such as optical filters, gratings, prisms, and chromatic beamsplitters.

In certain embodiments, light conditioning optics 2254 modify thespatial properties of the light generated by light source 2252. Examplesof components that can be used for this purpose include (but are notlimited to) apertures, phase masks, apodizing elements, and diffusers.

After modification by light conditioning optics 2254, the light isreceived by light delivery optics 2256 and directed onto sample 2208 orfeature array 2214, either of which is positioned on a mount 2258. Lightconditioning optics 2254 generally function to collect and direct lightonto the surface of the sample or array. A variety of different opticalelements can be used for this purpose, and examples of such elementsinclude, but are not limited to, lenses, mirrors, beam splitters, andvarious other elements having non-zero optical power.

Light emerging from sample 2208 or feature array 2214 is collected bylight collection optics 2260. In general, light collection optics 2260can include elements similar to any of those described above inconnection with light delivery optics 2256. The collected light can thenoptionally be modified by light adjusting optics 2262, which cangenerally include any of the elements described above in connection withlight conditioning optics 2254.

The light is then detected by detection sub-system 2264. Generally,detection sub-system 2264 functions to generate one or more images ofsample 2208 or feature array 2214 by detecting light from the sample orfeature array. A variety of different imaging elements can be used indetection sub-system 2264, including CCD detectors and other imagecapture devices.

Each of the foregoing components can optionally be connected to controlunit 2228 as shown in FIG. 22B, so that control unit 2228 can adjustvarious properties of the imaging apparatus. For example, control unit2228 can adjust the position of sample 2208 or feature array 2214relative to the position of the incident light, and also with respect tothe focal plane of the incident light (if the incident light isfocused). Control unit 2228 can also selectively filter both theincident light and the light emerging from the sample.

Imaging apparatus 2250 can typically obtain images in a variety ofdifferent imaging modalities. In some embodiments, for example, theimages are transmitted light images, as shown in FIG. 22B. In certainembodiments, apparatus 2250 is configured to obtain reflection images.In some embodiments, apparatus 2250 can be configured to obtainbirefringence images, fluorescence images, phosphorescence images,multiphoton absorption images, and more generally, any known image type.

In general, control unit 2228 can perform any of the method stepsdescribed herein that do not expressly require user intervention bytransmitting suitable control signals to the components of samplehandling apparatus 2200 and/or imaging apparatus 2250. To perform suchsteps, control unit 2228 generally includes software instructions that,when executed, cause control unit 2228 to undertake specific steps. Insome embodiments, control unit 2228 includes an electronic processor andsoftware instructions that are readable by the electronic processor, andcause the processor to carry out the steps describe herein. In certainembodiments, control unit 2228 includes one or more application-specificintegrated circuits having circuit configurations that effectivelyfunction as software instructions.

Control unit 2228 can be implemented in a variety of ways. FIG. 22C is aschematic diagram showing one example of control unit 2228, including anelectronic processor 2280, a memory unit 2282, a storage device 2284,and an input/output interface 2286. Processor 2280 is capable ofprocessing instructions stored in memory unit 2282 or in storage device2284, and to display information on input/output interface 2286.

Memory unit 2282 stores information. In some embodiments, memory unit2282 is a computer-readable medium. Memory unit 2282 can includevolatile memory and/or non-volatile memory. Storage device 2284 iscapable of providing mass storage, and in some embodiments, is acomputer-readable medium. In certain embodiments, storage device 2284may be a floppy disk device, a hard disk device, an optical disk device,a tape device, a solid state device, or another type of writeablemedium.

The input/output interface 2286 implements input/output operations. Insome embodiments, the input/output interface 2286 includes a keyboardand/or pointing device. In some embodiments, the input/output interface2286 includes a display unit for displaying graphical user interfacesand/or display information.

Instructions that are executed and cause control unit 2228 to performany of the steps or procedures described herein can be implemented indigital electronic circuitry, or in computer hardware, firmware, or incombinations of these. The instructions can be implemented in a computerprogram product tangibly embodied in an information carrier, e.g., in amachine-readable storage device, for execution by a programmableprocessor (e.g., processor 2280). The computer program can be written inany form of programming language, including compiled or interpretedlanguages, and it can be deployed in any form, including as astand-alone program or as a module, component, subroutine, or other unitsuitable for use in a computing environment. Storage devices suitablefor tangibly embodying computer program instructions and data includeall forms of non-volatile memory, including by way of examplesemiconductor memory devices, such as EPROM, EEPROM, and flash memorydevices; magnetic disks such as internal hard disks and removable disks;magneto-optical disks; and CD-ROM and DVD-ROM disks. The processor andthe memory can be supplemented by, or incorporated in, ASICs(application-specific integrated circuits).

Processor 2280 can include any one or more of a variety of suitableprocessors. Suitable processors for the execution of a program ofinstructions include, by way of example, both general and specialpurpose microprocessors, and the sole processor or one of multipleprocessors of any kind of computer or computing device.

Exemplary Embodiments

In some non-limiting examples of the workflows described herein, thesample can be immersed in 100% chilled methanol and incubated for 30minutes at -20° C. After 20 minutes, the sample can be removed andrinsed in ultrapure water. After rinsing the sample, fresh eosinsolution is prepared, and the sample can be covered in isopropanol.After incubating the sample in isopropanol for 1 minute, the reagent canbe removed by holding the slide at an angle, where the bottom edge ofthe slide can be in contact with a laboratory wipe and air dried. Thesample can be uniformly covered in hematoxylin solution and incubatedfor 7 minutes at room temperature. After incubating the sample inhematoxylin for 7 minutes, the reagent can be removed by holding theslide at an angle, where the bottom edge of the slide can be in contactwith a laboratory wipe. The slide containing the sample can be immersedin water and the excess liquid can be removed. After that, the samplecan be covered with blueing buffer and can be incubated for 2 minutes atroom temperature. The slide containing the sample can again be immersedin water, and uniformly covered with eosin solution and incubated for 1minute at room temperature. The slide can be air-dried for no more than30 minutes and incubated for 5 minutes at 37° C. The sample can beimaged using brightfield imaging setting.

Further, the biological sample can be processed by the followingexemplary steps for sample permeabilization and cDNA generation. Thesample can be exposed to a permeabilization enzyme and incubated at 37°C. for the pre-determined permeabilization time (which is tissue typespecific). The permeabilization enzyme can be removed and the sampleprepared for analyte capture by adding 0.1X SSC buffer. The sample canthen subjected to a pre-equilibration thermocycling protocol (e.g., lidtemperature and pre-equilibrate at 53° C., reverse transcription at 53°C. for 45 minutes, and then hold at 4° C.) and the SSC buffer can beremoved. A Master Mix, containing nuclease-free water, a reversetranscriptase reagent, a template switch oligo, a reducing agent, and areverse transcriptase enzyme can be added to the biological sample andsubstrate, and the sample with the Master Mix can be subjected to athermocycling protocol (e.g., perform reverse transcription at 53° C.for 45 minutes and hold at 4° C.). Second strand synthesis can beperformed on the substrate by subjecting the substrate to athermocycling protocol (e.g., pre-equilibrate at 65° C., second strandsynthesis at 65° C. for 15 minutes, then hold at 4° C.). The Master Mixreagents can be removed from the sample and 0.8 M KOH can be applied andincubated for 5 minutes at room temperature. The KOH can be removed andelution buffer can be added and removed from the sample. A Second StrandMix, including a second strand reagent, a second strand primer, and asecond strand enzyme, can be added to the sample and the sample can besealed and incubated. At the end of the incubation, the reagents can beremoved and elution buffer can be added and removed from the sample, and0.8 M KOH can be added again to the sample and the sample can beincubated for 10 minutes at room temperature. Tris-HCl can be added andthe reagents can be mixed. The sample can be transferred to a new tube,vortexed, and placed on ice.

Further the biological sample can be processed by the followingexemplary steps for cDNA amplification and quality control. A qPCR Mix,including nuclease-free water, qPCR Master Mix, and cDNA primers, can beprepared and pipetted into wells in a qPCR plate. A small amount ofsample can be added to the plated qPCR Mix, and thermocycled accordingto a predetermined thermocycling protocol (e.g., step 1: 98° C. for 3minutes, step 2: 98° C. for 5 seconds, step 3: 63° C. for 30 seconds,step 4: record amplification signal, step 5: repeating 98° C. for 5seconds, 63° C. for 30 seconds for a total of 25 cycles). Aftercompleting the thermocycling, a cDNA amplification mix, includingamplification mix and cDNA primers, can be prepared and combined withthe remaining sample and mixed. The sample can then be incubated andthermocycled (e.g., lid temperature at 105° C. for ~45-60 minutes; step1: 98° C. for 3 minutes, step 2: 98° C. for 15 seconds, step 3: 63° C.for 20 seconds, step 4: 72° C. for one minute, step 5: [the number ofcycles determined by qPCR Cq Values], step 6: 72° C. for 1 minute, andstep 7: hold at 4° C.). The sample can then be stored at 4° C. for up to72 hours or at -20° C. for up to 1 week, or resuspended in 0.6XSPRIselect Reagent and pipetted to ensure proper mixing. The sample canthen be incubated at 5 minutes at room temperature, and cleared byplacing the sample on a magnet (e.g., the magnet is in the highposition). The supernatant can be removed and 80% ethanol can be addedto the pellet, and incubated for 30 seconds. The ethanol can be removedand the pellet can be washed again. The sample can then be centrifugedand placed on a magnet (e.g., the magnet is on the low position). Anyremaining ethanol can be removed and the sample can be air dried for upto 2 minutes. The magnet can be removed and elution buffer can be addedto the sample, mixed, and incubated for 2 minutes at room temperature.The sample can then be placed on the magnet (e.g., on low position)until the solution clears. The sample can be transferred to a new tubestrip and stored at 4° C. for up to 72 hours or at -20° C. for up to 4weeks. A portion of the sample can be run on an Agilent Bioanalyzer HighSensitivity chip, where a region can be selected and the cDNAconcentration can be measured to calculate the total cDNA yield.Alternatively, the quantification can be determined by AgilentBioanalyzer or Agilent TapeStation.

Further, the biological sample can be processed by the followingexemplary steps for spatial gene expression library construction. AFragmentation Mix, including a fragmentation buffer and fragmentationenzyme, can be prepared on ice. Elution buffer and fragmentation mix canbe added to each sample, mixed, and centrifuged. The sample mix can thenbe placed in a thermocycler and cycled according to a predeterminedprotocol (e.g., lid temperature at 65° C. for ~ 35 minutes, pre-coolblock down to 4° C. before fragmentation at 32° C. for 5 minutes,End-repair and A-tailing at 65° C. for 30 minutes, and holding at 4°C.). The 0.6X SPRIselect Reagent can be added to the sample andincubated at 5 minutes at room temperature. The sample can be placed ona magnet (e.g., in the high position) until the solution clears, and thesupernatant can be transferred to a new tube strip. 0.8X SPRIselectReagent can be added to the sample, mixed, and incubated for 5 minutesat room temperature. The sample can be placed on a magnet (e.g., in thehigh position) until the solution clears. The supernatant can be removedand 80% ethanol can be added to the pellet, the pellet can be incubatedfor 30 seconds, and the ethanol can be removed. The ethanol wash can berepeated and the sample placed on a magnet (e.g., in the low position)until the solution clears. The remaining ethanol can be removed andelution buffer can be added to the sample, mixed, and incubated for 2minutes at room temperature. The sample can be placed on a magnet (e.g.,in the high position) until the solution clears, and a portion of thesample can be moved to a new tube strip. An Adaptor Ligation Mix,including ligation buffer, DNA ligase, and adaptor oligos, can beprepared and centrifuged. The Adaptor Ligation Mix can be added to thesample, pipette-mixed, and centrifuged briefly. The sample can then bethermocycled according to a predetermined protocol (e.g., lidtemperature at 30° C. for ~15 minutes, step 1: 20° C. for 15 minutes,step 2: 4° C. hold). The sample can be vortexed to re-suspend SPRIselectReagent, additional 0.8X SPRIselect Reagent can be added to the sampleand incubated for 5 minutes at room temperature, and placed on a magnet(e.g., in the high position) until the solution clears. The supernatantcan be removed and the pellet can be washed with 80% ethanol, incubatedfor 30 seconds, and the ethanol can be removed. The ethanol wash can berepeated, and the sample can be centrifuged briefly before placing thesample on a magnet (e.g., in the low position). Any remaining ethanolcan be removed and the sample can be air dried for a maximum of 2minutes. The magnet can be removed, and elution buffer can be added tothe sample, and the sample can be pipette-mixed, incubated for 2 minutesat room temperature, and placed on a magnet (e.g., in the low position)until the solution clears. A portion of the sample can be transferred toa new tube strip. Amplification mix, can be prepared and combined withthe sample. An individual Dual Index TT Set A can be added to thesample, pipette-mixed and subjected to a pre-determined thermocyclingprotocol (e.g., lid temperature at 105° C. for ~25-40 minutes, step 1:98° C. for 45 seconds, step 2: 98° C. for 20 seconds, step 3: 54° C. for30 seconds; step 4: 72° C. for 20 seconds, step 5: reverting to step 2for a predetermined number of cycles, step 6: 72° C. for 1 minute, and4° C. on hold). Vortex to re-suspend the SPRIselect Reagent, additional0.6X SPRIselect Reagent can be added to each sample, mixed, andincubated for 5 minutes at room temperature. The sample can be placed ona magnet (e.g., in the high position) until the solution clears, and thesupernatant can be transferred to a new tube strip. The 0.8X SPRIselectReagent can be added to each sample, pipette-mixed, and incubated for 5minutes at room temperature. The sample can then be placed on a magnet(e.g., in the high position) until the solution clears. The supernatantcan be removed, and the pellet can be washed with 80% ethanol, incubatedfor 30 seconds, and then the ethanol can be removed. The ethanol washcan be repeated, the sample centrifuged, and placed on a magnet (e.g.,in the low position) to remove any remaining ethanol. The sample can beremoved from the magnet and Elution Buffer can be added to the sample,pipette-mixed, and incubated at 2 minutes at room temperature. Thesample can be placed on a magnet (e.g., in the low position) until thesolution clears and a portion of the sample can be transferred to a newtube strip. The sample can be stored at 4° C. for up to 72 hours, or at-20° C. for long-term storage. The average fragment size can bedetermined using a Bioanalyzer trace or an Agilent TapeStation.

The library can be sequenced using available sequencing platforms,including, MiSeq, NextSeq 500/550, HiSeq 2500, HiSeq 3000/4000, NovaSeq,and iSeq.

In alternate embodiments of the above described workflows, a biologicalsample can be permeabilized by exposing the sample to greater than about1.0 w/v % (e.g., greater than about 2.0 w/v %, greater than about 3.0w/v %, greater than about 4.0 w/v%, greater than about 5.0 w/v %,greater than about 6.0 w/v %, greater than about 7.0 w/v %, greater thanabout 8.0 w/v %, greater than about 9.0 w/v %, greater than about 10.0w/v %, greater than about 11.0 w/v %, greater than about 12.0 w/v %, orgreater than about 13.0 w/v %) sodium dodecyl sulfate (SDS). In someembodiments, a biological sample can be permeabilized by exposing thesample (e.g., for about 5 minutes to about 1 hour, about 5 minutes toabout 40 minutes, about 5 minutes to about 30 minutes, about 5 minutesto about 20 minutes, or about 5 minutes to about 10 minutes) to about1.0 w/v % to about 14.0 w/v % (e.g., about 2.0 w/v % to about 14.0 w/v%, about 2.0 w/v % to about 12.0 w/v %, about 2.0 w/v % to about 10.0w/v %, about 4.0 w/v % to about 14.0 w/v %, about 4.0 w/v % to about12.0 w/v %, about 4.0 w/v % to about 10.0 w/v %, about 6.0 w/v % toabout 14.0 w/v %, about 6.0 w/v % to about 12.0 w/v %, about 6.0 w/v %to about 10.0 w/v %, about 8.0 w/v % to about 14.0 w/v %, about 8.0 w/v% to about 12.0 w/v %, about 8.0 w/v % to about 10.0 w/v %, about 10.0 %w/v % to about 14.0 w/v %, about 10.0 w/v % to about 12.0 w/v %, orabout 12.0 w/v % to about 14.0 w/v %) SDS and/or proteinase K (e.g., ata temperature of about 35° C. to about 50° C., about 35° C. to about 45°C., about 35° C. to about 40° C., about 40° C. to about 50° C., about40° C. to about 45° C., or about 45° C. to about 50° C.).

In non-limiting examples of any of the workflows described herein, anucleic acid molecule is produced that includes a contiguous nucleotidesequence comprising: (a) a first primer sequence (e.g., Read 1); (b) aspatial barcode; (c) a unique molecular sequence (UMI); (d) a capturedomain; (e) a sequence complementary to a sequence present in a nucleicacid from a biological sample; (f) a second primer sequence (e.g., Read2) that is substantially complementary to a sequence of a templateswitching oligonucleotide (TSO). In some embodiments of these nucleicacid molecules, the nucleic acid molecule is a single-stranded nucleicacid molecule. In some embodiments of these nucleic acid molecules, thenucleic acid molecule is a double-stranded nucleic acid molecule. Insome embodiments of these nucleic acid molecules, (a) through (f) arepositioned in a 5′ to 3′ direction in the contiguous nucleotidesequence. In some embodiments of any of these nucleic acid molecules,the nucleic acid molecule is attached to a substrate (e.g., a slide). Insome embodiments of any of these nucleic acid molecules, the 5′ end ofthe contiguous nucleic acid sequence is attached to the substrate (e.g.,a slide). In some embodiments of any of these nucleic acid molecules,the contiguous nucleotide sequence is a chimeric RNA and DNA sequence.In some embodiments of any of these nucleic acid molecules, thecontiguous nucleotide sequence is a DNA sequence.

In non-limiting examples of any of the workflows described herein, anucleic acid molecule is produced that includes a contiguous nucleotidesequence comprising: (a) a sequence complementary to a first primersequence (e.g., a sequence complementary to Read 1); (b) a sequencecomplementary to a spatial barcode; (c) a sequence complementary to aunique molecular sequence; (d) a sequence complementary to a capturedomain; (e) a sequence present in a nucleic acid from a biologicalsample; and (f) a sequence of a template switching oligonucleotide(TSO). In some embodiments of any of these nucleic acid molecules, thenucleic acid molecule is single-stranded. In some embodiments of any ofthese nucleic acid molecules, the nucleic acid molecule isdouble-stranded. In some embodiments of any of these nucleic acidmolecules, the contiguous nucleotide sequence is a DNA sequence. In someembodiments of any of these nucleic acid molecules, (a) through (f) arepositioned in a 3′ to 5′ direction in the contiguous nucleotidesequence.

In non-limiting examples of any of the workflows described herein, anucleic acid molecule is produced that includes a contiguous nucleotidesequence comprising: (a) a first primer sequence (e.g., Read 1); (b) aspatial barcode; (c) a unique molecular sequence (UMI); (d) a capturedomain; (e) a sequence complementary to a sequence present in a nucleicacid from a biological sample; and (f) a second primer sequence (Read2). In some embodiments of any of these nucleic acid molecules, thenucleic acid molecule is a single-stranded nucleic acid molecule. Insome embodiments of any of these nucleic acid molecules, the nucleicacid molecule is a double-stranded nucleic acid molecule. In someembodiments of any of these nucleic acid molecules, (a) through (f) arepositioned in a 5′ to 3′ direction in the contiguous nucleotidesequence. In some embodiments of any of these nucleic acid molecules,the contiguous nucleotide sequence is a DNA sequence. In someembodiments of any of these nucleic acid molecules, the contiguousnucleotide sequence further comprises 3′ to (f): (g) a sequencecomplementary to a first adaptor sequence; and (h) a sequencecomplementary to a third primer sequence. In some embodiments of any ofthe nucleic acid molecules, the first adaptor sequence is an i7 sampleindex sequence. In some embodiments of any of these nucleic acidmolecules, the third primer sequence is a P7 primer sequence. In someembodiments of any of these nucleic acid molecules, (h) is 3′ positionedrelative to (g) in the contiguous nucleotide sequence. In someembodiments of any of these nucleic acid molecules, the contiguousnucleotide sequence further comprises 5′ to (a): (i) a second adaptorsequence; and (ii) a fourth primer sequence. In some embodiments of anyof these nucleic acid molecules, the second adaptor sequence is an i5sample index sequence. In some embodiments of any of these nucleic acidmolecules, the fourth primer sequence is a P5 primer sequence. In someembodiments of any of these nucleic acid molecules, (ii) is 5′positioned relative to (i) in the contiguous nucleotide sequence.

In non-limiting examples of any of the workflows described herein, anucleic acid molecule is produced that includes a contiguous nucleotidesequence comprising: (a) a sequence complementary to a first primersequence; (b) a sequence complementary to a spatial barcode; (c) asequence complementary to a unique molecular sequence; (d) a sequencecomplementary to a capture domain; (e) a sequence present in a nucleicacid from a biological sample; and (f) a sequence complementary to asecond primer sequence. In some embodiments of these nucleic acidmolecules, a sequence complementary to a first primer sequence is asequence complementary to Read 1. In some embodiments of these nucleicacid molecules, a sequence complementary to a second primer sequence isa sequence complementary to Read 2. In some embodiments of any of thesenucleic acid molecules, the nucleic acid molecule is a single-strandednucleic acid molecule. In some embodiments of any of these nucleic acidmolecules, the nucleic acid molecule is a double-stranded nucleic acidmolecule. In some embodiments of any of these nucleic acid molecules,(a) through (f) are positioned in a 3′ to 5′ direction in the contiguousnucleotide sequence. In some embodiments of any of these nucleic acidmolecules, the contiguous nucleotide sequence is a DNA sequence. In someembodiments of any of these nucleic acid molecules, the contiguousnucleotide sequence further comprises 5′ to (f): (g) a first adaptorsequence; and (h) a third primer sequence. In some embodiments of any ofthese nucleic acid molecules, the first adaptor sequence is an i7 sampleindex sequence. In some embodiments of any of these nucleic acidmolecules, the third primer sequence is a P7 primer sequence. In someembodiments of any of these nucleic acid molecules, (h) is 5′ positionedrelative to (g) in the contiguous nucleotide sequence. In someembodiments of any of these nucleic acid molecules, the contiguousnucleotide sequence further comprises 3′ to (a): (i) a sequencecomplementary to a second adaptor sequence; and (ii) a sequencecomplementary to a fourth primer sequence. In some embodiments of any ofthese nucleic acid molecules, the second adaptor sequence is an i5sample index sequence. In some embodiments of any of these nucleic acidmolecules, the fourth primer sequence is a P5 primer sequence. In someembodiments of any of these nucleic acid molecules, (ii) is 3′positioned relative to (i) in the contiguous nucleotide sequence.

Spatial RIN

Provided herein is a non-limiting example of a protocol for determiningthe spatial RIN in a tissue that can include collecting breast cancertissue and snap freezing in liquid nitrogen. Tissue can be embedded inOCT and sectioned at 10 or 12 µM thickness at -20° C. and mounteddirectly on a spatial array including capture probes having an 18S rRNAcapture domain. Tissue can be fixed and stained using a Hematoxylin andEosin (H&E) staining protocol. Briefly, Mayer’s Hematoxylin can beadded, washed in water, incubated in Bluing buffer, washed in water,stained with Eosin, then washed in water, and finally dried. Forvisualizing H&E staining, sections can be mounted with 85% glycerol andcovered with a coverslip. Bright field imaging can be performed usingthe Metafer Slide Scanning Platform (Metasystems) where raw images arestitched together with the VSlide software (Metasystems). Glycerol canbe removed by holding the spatial array or glass slide in water untilthe coverslip falls off and then was air dry until the remaining liquidevaporates. Hematoxylin, from the H&E stain, can be optionally removedfrom the tissue section, for example, by washing in dilute HCl (0.01 M)prior to further processing. The tissue sections are then ready forfurther processing.

Following staining, the 18S rRNA present in the tissue sections arecaptured by the 18S rRNA specific capture domains on the spatial array.The 18S rRNA is then converted to cDNA in situ. Specifically, reversetranscription is performed on the spatial array in a sealedhybridization cassette by adding 70µl reaction mixture including 1xFirst-strand buffer, 5 mM DTT, 1 M Betaine, 6 mM MgCl2, 1 mM dNTPs, 0.2mg/ml BSA, 50 ng/µl Actinomycin D, 10% DMSO, 20 U/µl SuperScript IIIReverse Transcriptase, 2 U/µl RNaseOUT Recombinant RibonucleaseInhibitor. The reaction is performed overnight at 42° C. overnight.After incubation cDNA synthesis mixture is removed and the tissue waswashed with 0.1xSSC buffer.

In order to prepare the spatial array for oligonucleotide probe labelingand imaging, the breast cancer tissue and rRNA is removed. Tissueremoval can be performed first by incubation with β-mercaptoethanol inRLT lysis buffer at a 3:100 ratio at 56° C. for 1 hour with continuousshaking at 300 rpm. All tissues can be incubated with a 1:7 ratio ofProteinase K and PKD buffer for 1 hour at 56° C. using short intervalswith gentle shaking at 300 rpm. The spatial array is then washed withcontinuous shaking at 300 rpm as follows: first in 2xSSC with 0.1% SDSat 50° C. for 10 min, then in 0.2xSSC at RT for 1 min and finally in0.1xSSC at RT for 1 min. The spatial array is then spin-dried and putback into the hybridization cassette. rRNA removal can be performedusing a reaction mixture containing the following final concentrations:1x First-strand buffer, 0.4 mg/ml BSA and 16.3 mU/µl RNase H. Thereaction can be performed for 1 hour at 37° C. with gentle shaking at300 rpm using short intervals. Spatial arrays are then washed with0.1xSSC buffer and treated with 60% DMSO at room temperature for 5minutes and then washed three times with 0.1xSSC buffer.

In order to detect the cDNA produced from the 18S rRNA, labeledoligonucleotide probes are generated that had sequence complementarityto the 18S cDNA sequence. Oligonucleotides are identified that have alength between 18-23 nucleotides with an optimum at 20 nucleotides, amelting temperature (Tm) between 38-50° C. with the optimal temperatureat 42° C., and a content of guanine and cytosine of 30-60% with anoptimum at 50%. The first five bases of the 3′-ends are set to includetwo of either guanine or cytosine or one of each. Oligonucleotides arechecked for quality using Mfold (determination of secondary structure),Oligo Calc: Oligonucleotide Properties Calculator (determination ofself-dimerization and hairpin formation), and BLAST (determination ofoff-target binding). The sequence locations are picked for compatibilitywith both human (NR _003286.2) and mouse (NR_003278.3) 18S rRNA. Four ofthe oligonucleotide probes selected include: probe 1 (P1; SEQ ID NO: 4)GAGGAATTCCCAGTAAGT, probe 2 (P2; SEQ ID NO: 5) GAGATTGAGCAATAACAG, probe3 (P3; SEQ ID NO: 6) GTAGTTCCGACCATAAAC, and probe 4 (P4; SEQ ID NO: 7)GGTGACTCTAGATAACCT. Control oligonucleotide probes can be designed toinclude complementary sequences of three detection probes at a time witha 20 bases spacer sequence between each probe. The selectedoligonucleotide probes can be then designed to incorporate a Cy3fluorophore.

Next, labeled oligonucleotide probes are hybridized to the spatial arraycontaining cDNA produced from the 18S rRNA or containing control captureprobes. This step can include at least 4 successive rounds ofhybridization and imaging, with at least one round for each of the fourlabeled oligonucleotide probes. Following each round of hybridizationand imaging, the spatial array can be washed to remove the hybridizedprobe before continuing with a subsequent round of hybridization andimaging.

Hybridization of labeled oligonucleotide probes includes adding to thespatial arrays a pre-heated (e.g., heated to 50° C.) hybridizationmixture (10 mM Tris-HCl, 1 mM EDTA, 50 mM

NaCl, and 0.5 µM of fluorescently labelled probe) containing at least0.5 µM of one of the fluorescently labeled oligonucleotide probes (e.g.,one of P1, P2, P3 or P4). The spatial array can be then imaged using aDNA microarray scanner with the following settings: excitationwavelength 532 nm set to gain 70 and 635 nm set to 1. Following imaging,the spatial array is incubated with 60% DMSO at room temperature for 5minutes and washed three times with 0.1xSSC buffer to remove thehybridized probe. Subsequent rounds of hybridization and imaging areperformed with a different labeled oligonucleotide probe used in eachround (e.g., round 2 used P2, round 3 used P3, and round 4 used P4). Aninitial, pre-hybridization round (P0) of imaging are performed in orderto assess background fluorescence.

One image is generated per labeled oligonucleotide probe (P1—P4) andalso one where no fluorescently labeled probes were hybridized (P0).Normalization of Fluorescence Units (FU) data is done by subtraction ofthe auto-fluorescence recorded with P0 and division with P1. Afteraligning the five images for a particular location in the tissue (oneimage from each probe, P1—P4, and one image from the location withoutlabeling), the images are loaded into a script and run in RStudio. TheScript can generate two different plots, one heat-map of spatial RINvalues and one image alignment error plot.

High quality RNA is defined as full-length (or close to full-length)transcripts, whereas low quality RNA is defined as fragmentedtranscripts. Spatial RIN values are similar to traditional RIN values inthat the RIN values range from 1 to 10, with higher numbers indicatinghigher quality (e.g., less degraded, less fragmented) RNA samples.

Spatial ATAC Compositions

Provided herein are compositions for identifying the location of ananalyte in a biological sample. In some embodiments, a nucleotidemolecule composition including a) a spatial barcode b) a unique moleculec) capture domain; d) a functional domain; and e) a splintoligonucleotide. In some embodiments, a partially double-strandednucleotide molecule composition including: a)a spatial barcode; b) aunique molecular identifier; c) a capture domain; d) a functionaldomain; and e) a splint oligonucleotide. In some embodiments, acomposition including a) a capture probe including i) a spatial barcode;ii) a unique molecular identifier; iii) a capture domain; iv) afunctional domain; and v) a splint oligonucleotide and b) fragmentedgenomic DNA including i) a first adapter sequence comprising atransposon end sequence and a sequence complementary to the capturedomain; and ii) a second adapter sequence comprising the transposon endsequence and a second adapter sequence. In some embodiments, acomposition including a) a transposase enzyme monomer complexed with afirst adapter including i) a transposon end sequence; and ii) a sequencecomplementary to the capture domain; and b) a transposase enzyme secondmonomer complexed with a second adapter including i) a transposon endsequence; and ii) a second adapter sequence; c) genomic DNA; and d) acapture probe, including i) a spatial barcode; ii) a unique molecularidentifier; iii) a capture domain; iv) a functional domain; and v) asplint oligonucleotide. In some embodiments, a composition including a)a transposase enzyme monomer complexed with a first adapter including i)a transposon end sequence; ii) a sequence complementary to the capturedomain; wherein the 5′ end of the first adapter is phosphorylated and b)a transposase enzyme second monomer complexed with a second adapterincluding i) the transposon end sequence; ii) a second adapter sequence;wherein the 5′ end of the second adapter is phosphorylated; c) genomicDNA; and d) a capture probe including i) a spatial barcode; ii) a uniquemolecular identifier; iii) a capture domain; iv) a functional domain;and v) a splint oligonucleotide. In some embodiments, a compositionincluding a) a transposase enzyme dimer including i) a transposaseenzyme monomer complexed with a first adapter including 1) a transposonend sequence, 2) a sequence complementary to a capture domain; ii) atransposase enzyme second monomer complexed with a second adapterincluding 1) the transposon end sequence; 2) a second adapter sequence;b) genomic DNA; c) a capture probe, including i) a spatial barcode; ii)a unique molecular identifier; iii) a capture domain; iv) a functionaldomain; and v) a splint oligonucleotide. In some embodiments, acomposition including a) a transposase enzyme dimer including i) atransposase enzyme monomer complexed with a first adapter including 1) atransposon end sequence, 2)a sequence complementary to a capture domainwherein the 5′ end of the first adapter is phosphorylated; ii) atransposase enzyme second monomer complexed with a second adapterincluding 1) the transposon end sequence; 2) a second adapter sequencewherein the 5′ end of the second adapter is phosphorylated; c) genomicDNA; and d) a capture probe, including i) a spatial barcode; ii) aunique molecular identifier; iii) a capture domain; iv) a functionaldomain; and v) a splint oligonucleotide.

Spatial Transcriptomics

In some embodiments, provided herein are methods for spatially detectingan analyte (e.g., detecting the location of an analyte, e.g., abiological analyte) from a biological sample (e.g., present in abiological sample such as a tissue section) that include: (a) providinga biological sample on a substrate; (b) staining the biological sampleon the substrate, imaging the stained biological sample, and selectingthe biological sample or subsection of the biological sample to subjectto spatial analysis; (c) providing an array comprising one or morepluralities of capture probes on a substrate; (d) contacting thebiological sample with the array, thereby allowing a capture probe ofthe one or more pluralities of capture probes to capture the biologicalanalyte of interest; and (e) analyzing the captured biological analyte,thereby spatially detecting the biological analyte of interest. Anyvariety of staining and imaging techniques as described herein or knownin the art can be used in accordance with methods described herein. Insome embodiments, the staining includes optical labels as describedherein, including, but not limited to, fluorescent, radioactive,chemiluminescent, calorimetric, or colorimetric detectable labels. Insome embodiments, the staining includes a fluorescent antibody directedto a target analyte (e.g., cell surface or intracellular proteins) inthe biological sample. In some embodiments, the staining includes animmunohistochemistry stain directed to a target analyte (e.g., cellsurface or intracellular proteins) in the biological sample. In someembodiments, the staining includes a chemical stain such as hematoxylinand eosin (H&E) or periodic acid-schiff (PAS). In some embodiments,significant time (e.g., days, months, or years) can elapse betweenstaining and/or imaging the biological sample and performing spatialtranscriptomic analysis. In some embodiments, reagents for performingspatial analysis are added to the biological sample before,contemporaneously with, or after the array is contacted to thebiological sample. In some embodiments, step (d) includes placing thearray onto the biological sample. In some embodiments, the array is aflexible array where the plurality of spatially-barcoded features (e.g.,capture probes) are attached to a flexible substrate. In someembodiments, measures are taken to slow down a reaction (e.g., coolingthe temperature of the biological sample or using enzymes thatpreferentially perform their primary function at lower or highertemperature as compared to their optimal functional temperature) beforethe array is contacted with the biological sample. In some embodiments,step (e) is performed without bringing the biological sample out ofcontact with the array. In some embodiments, step (e) is performed afterthe biological sample is no longer in contact with the array. In someembodiments, the biological sample is tagged with an analyte captureagent before, contemporaneously with, or after staining and/or imagingof the biological sample. In such cases, significant time (e.g., days,months, or years) can elapse between staining and/or imaging andperforming spatial analysis. In some embodiments, the array is adaptedto facilitate biological analyte migration from the stained and/orimaged biological sample onto the array (e.g., using any of thematerials or methods described herein). In some embodiments, abiological sample is permeabilized before being contacted with an array.In some embodiments, the rate of permeabilization is slowed prior tocontacting a biological sample with an array (e.g., to limit diffusionof analytes away from their original locations in the biologicalsample). In some embodiments, modulating the rate of permeabilization(e.g., modulating the activity of a permeabilization reagent) can occurby modulating a condition that the biological sample is exposed to(e.g., modulating temperature, pH, and/or light). In some embodiments,modulating the rate of permeabilization includes use of external stimuli(e.g., small molecules, enzymes, and/or activating reagents) to modulatethe rate of permeabilization. For example, a permeabilization reagentcan be provided to a biological sample prior to contact with an array,which permeabilization reagent is inactive until a condition (e.g.,temperature, pH, and/or light) is changed or an external stimulus (e.g.,a small molecule, an enzyme, and/or an activating reagent) is provided.

Spatially-Resolved Gene Expression and Clustering in Invasive DuctalCarcinoma

The spatial gene expression of invasive ductal carcinoma tissue from afemale patient (ER+, PR-, HER2+) was profiled (BioIVT: Asterand - CaseID 66320; Specimen ID 116899F). As a control, the healthy tissuesections adjacent to the tumor were obtained. 4 replicates were used foreach tissue type.

Spatially-resolved gene expression and clustering in invasive ductalcarcinoma reveal intra-tumor heterogeneity is shown in FIGS. 23A-H. FIG.23A shows a histological section of an invasive ductal carcinomaannotated by a pathologist. The section contains a large proportion ofinvasive carcinoma (22.344 mm² portion indicated by thick black line(outlined in black in color figure)), three separate ductal cancer insitu regions (portions indicated by medium thickness black line 1.329mm², 1.242 mm², and 0.192 mm² (outlined in green in color figure)), andfibrous tissue. FIG. 23B shows a tissue plot with spots colored byunsupervised clustering of transcripts. FIG. 23C shows a t-SNE plot ofspots colored by unsupervised clustering of transcripts. FIG. 23D showsa gene expression heat map of the most variable genes between the 9identified clusters. The region defined as fibrous tissue mostlycorresponds to clusters 1, 7, and 8. Interestingly, a large regionannotated as invasive carcinoma by a pathologist contained spatial spotsthat were assigned to DCIS (cluster 5). In addition, four subtypes ofinvasive carcinoma with distinct molecular properties (clusters 2, 3, 4,and 6) were identified, revealing intra-tumor heterogeneity.

The expression levels of genes corresponding to human epidermal growthfactor receptor 2 (Her2), estrogen receptor (ER), and progesteronereceptor (PGR) in the tissue section are shown in FIG. 23E. It isclearly visible that Her2 and ER are highly expressed in the invasivecarcinoma and DCIS regions while the expression of PR is absent,consistent with the patient’s diagnosis. One of the top differentiallyexpressed genes from each cluster in the invasive carcinoma region wasselected (rectangular boxes in FIG. 23D), and its expression levels arelocated in the tissue as shown in FIG. 23F and overlapped in one plot asshown in FIG. 23G. With the exception of PGR, all of these genes werehighly up-regulated in the carcinoma tissue compared to the adjacentnormal tissue (FIG. 23H). Analysis revealed that all of theseup-regulated genes have implication in cancer progression.Interestingly, in the subset of cluster 3, a long non-coding RNA, ofwhich abnormal expression has recently been implicated in tumordevelopment (see, e.g., Zhang T, et al. Long Non-Coding RNA and BreastCancer. Technol Cancer Res Treat. 2019, 18,1533033819843889,incorporated herein by reference in its entirety), is one of the topdifferentially expressed genes. In glioblastoma, LINC00645 promotesepithelial-to-mesenchymal transition by inducing TGF-β (see, e.g., Li,C. et al. Long non-coding RNA linc00645 promotes TGF-β-inducedepithelial-mesenchymal transition by regulating miR-205-3p-ZEB1 axis inglioma. Cell Death & Dis. 2019, 10, 272, incorporated herein byreference in its entirety).

During breast cancer progression, the myoepithelial cells, whichcontinue to surround preinvasive in situ carcinoma, gradually disappear(see, e.g., Gudjonsson, T. et al. Myoepithelial Cells: Their Origin andFunction in Breast Morphogenesis and Neoplasia. J. Mammary Gland Biol.Neoplasia. 2009, 10, 261, incorporated herein by reference in itsentirety). This phenomenon is clearly visualized in FIGS. 23I and 23Jwhere KRT14 (a gene signature of myoepithelial cells) was highlyexpressed around the lining of the duct in the normal tissue while itwas disappearing in the DCIS region in IDC tissue (FIG. 23I). Theextracellular matrix genes such as COL1A1 and FN1, key genes associatedwith invasion and metastasis, were highly upregulated while smoothmuscles and basal keratin were downregulated in IDC (FIG. 23J).

VI. Generation of Spatial Arrays

In some aspects, provided herein are methods for generating arrays forspatial analysis (e.g., spatial arrays). The spatial arrays can havecapture probes that can interact with analytes present in a biologicalsample either directly or indirectly. For example, analytes can be RNA,DNA, protein, lipids, or any other analytes described herein. In someembodiments, the capture probe is a nucleic acid or a polypeptide. Insome embodiments, the capture probe is a conjugate (e.g., anoligonucleotide-antibody conjugate). In some embodiments, the captureprobe includes a barcode (e.g., a spatial barcode and/or a uniquemolecular identifier (UMI)) and a capture domain. In some embodiments, acapture probe includes a cleavage domain.

In some embodiments, capture probes are attached to an array feature andan individual array feature can include one or more capture probes. Insome embodiments, an individual array feature includes hundreds orthousands of capture probes. In some embodiments, the capture probes areassociated with a particular individual feature, where the individualfeature contains a capture probe including a spatial barcode unique tothe individual feature (e.g., a defined region or location on thearray).

In some embodiments, a particular feature can contain capture probesincluding more than one spatial barcode (e.g., one capture probe at aparticular feature can include a non-common spatial barcode that isdifferent than the non-common spatial barcode included in anothercapture probe at the same particular feature, while both capture probesinclude a second, common spatial barcode), where the non-common spatialbarcodes of the capture probes corresponds to a particular definedregion or location on the array. For example, multiple non-commonspatial barcode sequences associated with one particular feature on anarray can provide a stronger address or attribution to a given locationby providing duplicate or independent confirmation of the location. Insome embodiments, the multiple non-common spatial barcodes representincreasing specificity of the location of the particular array point

As used herein, spatial analyte capture can utilize nucleic acid probes(e.g., capture probes) including spatial barcode sequences, which areimmobilized on a substrate (e.g., directly or indirectly) to generate anarray. The spatial barcode sequences can correspond to the location ofthe capture probes on the array. In some embodiments, biologicalsamples, such as tissue sections, are placed onto the array and ananalyte from the biological sample is captured. In some embodiments, thecaptured analyte is a protein. In some embodiments, the captured analyteis a nucleic acid. In some embodiments, the captured analyte is RNA(e.g. messenger RNA). In some embodiments, the captured analyte is DNA(e.g., genomic DNA). In some embodiments, DNA from the biological sampleis processed, e.g. fragmented and/or provided with a binding domain thathybridizes to the capture probes on the array. In some embodiments, DNApolymerase extension or ligation reaction is performed in the biologicalsample on the array. In some embodiments, the probes to which nucleicacids in the biological sample bind are extended using the bound nucleicacid as a template to generate complementary nucleic acid strands, whichis immobilized on the surface of the array. In some embodiments,extended probes (e.g., capture probes that are subjected totemplate-directed nucleic acid polymerization using a captured nucleicacid analyte as a template) carry information about the position of the“captured” analytes in the biological sample, e.g. tissue section. Insome embodiments, the extended probes can be released from the surfaceof the array and analyzed, e.g. sequenced, which results in a “library”of captured nucleic acid analytes that are associated with spatialinformation. In some embodiments, the sequence data can then be matchedto a location in the biological sample, e.g. tissue section. Forinstance, the biological sample can be visualized or imaged, e.g.,stained and/or photographed, before or after analyte capture tofacilitate the correlation of the spatial barcode in the capture probe(e.g., an extended capture probe that is associated with a givenanalyte) with a position within the biological sample and the sequencedata can be overlaid on an image of the biological sample, e.g., using acomputer, to display the distribution pattern of any analyte of interestacross the biological sample. The visualization step is not essential asthe tagged analytes can be correlated with a location in the biologicalsample using other means, e.g., the unique profile of the analytescaptured with the same spatial barcode, e.g., at the same position onthe array, can enable the analyte to be correlated to a location withinthe biological sample based on the characteristics of cells or areas ofcells within the biological sample (e.g., a tissue section). Any of avariety of analytes can be captured and analyzed in accordance withmethods provided herein, including but not limited to RNA, specificsubtypes of RNA, proteins, antibodies, genomic DNA, T-cell receptors,small molecules, or other analytes.

Capture Probes via Splint Oligonucleotides

Capture probes for spatial analysis can be generated by differentmethods. In some embodiments, the capture probe is generated from two ormore oligonucleotide sequences. In some embodiments, two or moreoligonucleotide sequences are ligated together to generate a captureprobe. In some embodiments, capture probe generation can be facilitatedby a splint oligonucleotide. In some embodiments, a plurality ofoligonucleotides are attached to a feature on the substrate. In someembodiments, the oligonucleotides of the array can be about 20 to about80 nucleotides long. In some embodiments, the oligonucleotides of thearray can be about 30 to about 70 nucleotides long. In some embodiments,the oligonucleotides of the array can be about 40 to about 60nucleotides long. In some embodiments, the oligonucleotides of the arraycan be about 45 to about 55 nucleotides long. In some embodiments, thecapture probe includes one or more constant sequences (e.g., any of thevariety of constant sequences described herein). For example, theoligonucleotide can have a functional domain (e.g. any of the variety offunctional domains described herein) and/or a cleavage domain (e.g. anyof the variety of cleavage domains described herein).

In some embodiments, a primer can hybridize to an oligonucleotideattached, directly or indirectly to the substrate (e.g., attached to afeature attached to a substrate, e.g., attached via a linker). In someembodiments, the oligonucleotides can be attached, directly orindirectly, to the substrate at the 3′ end, resulting in the attachingoligonucleotides having free 5′ ends. For example, an array including aplurality of oligonucleotides attached to a substrate at their 3′ endscan be contacted with a plurality of primers. In some embodiments, theplurality of primers are complementary or substantially complementary tothe oligonucleotides attached to the substrate at their 3′ ends, or aportion thereof. In some embodiments the primer can be extended usingthe oligonucleotide attached to the substrate as a template to generatea first oligonucleotide. In some embodiments, the primer is extended bya reverse transcriptase enzyme. In some embodiments, the primer isextended by a DNA polymerase enzyme. In some embodiments, additionalnucleotides can be added (e.g., added by an enzyme) to the end of thefirst oligonucleotide to generate a 3′ overhang. In some embodiments,the enzyme is a template independent polymerase (e.g., any of thevariety of template independent polymerases described herein orotherwise known in the art). In some embodiments, the templateindependent polymerase is terminal nucleotidyl transferase. In someembodiments, the template independent polymerase is a poly(A)polymerase.

In some embodiments, oligonucleotides attached directly or indirectly tothe substrate at the 3′ end and having free 5′ ends can include asequence that indicates the position of a given oligonucleotide on thesubstrate. In some embodiments, such a sequence is or can function as aspatial barcode. In some embodiments, different oligonucleotidesattached directly or indirectly to the substrate at the 3′ end andhaving free 5′ ends can include different sequences that can indicatethe position of the different oligonucleotide on the substrate (e.g.,the different oligonucleotides can have different spatial barcodes orsequences that act as different spatial barcodes).

In some embodiments, the first oligonucleotide including the 3′ overhanggenerated by the template independent polymerase can be contacted with asplint oligonucleotide. The term “splint oligonucleotide”, as usedherein, refers to an oligonucleotide that, when hybridized to otherpolynucleotides, acts as a “splint” to position the polynucleotides(e.g., oligonucleotides) next to one another (e.g., abutting oneanother) so that they can be ligated together. In some embodiments, thesplint oligonucleotide is DNA. In some embodiments, the splintoligonucleotide is RNA. In some embodiments, the splint oligonucleotideincludes a nucleotide sequence that is partially complementary tonucleotide sequences from two or more different oligonucleotides. Insome embodiments, the splint oligonucleotide is approximately 40nucleotides in length. In some embodiments, the splint oligonucleotideis between about 10 and about 50 oligonucleotides in length. In someembodiments, the splint oligonucleotide is between about 10 and about45, about 10 and about 40, about 10 and about 35, about 10 and about 30,about 10 and about 25, or about 10 and about 20 oligonucleotides inlength. In some embodiments, the splint oligonucleotide is between about15 and about 50, about 15 and about 45, about 15 and about 40, about 15and about 35, about 15 and about 30, about 15 and about 30, or about 15and about 25 nucleotides in length.

In some embodiments, the splint oligonucleotide provided to the firstoligonucleotides can have a sequence that is complementary to thenucleotides of a generated 3′ overhang of the first oligonucleotide. Asecond oligonucleotide can be provided to the array. In someembodiments, the splint oligonucleotide can have another sequence thatis complementary or substantially complementary to the secondoligonucleotide, or portion thereof. In some embodiments, the splintoligonucleotide can hybridize to both the 3′ overhang of the firstoligonucleotide and the second oligonucleotide at the same time. In someembodiments, the first oligonucleotide and the second oligonucleotideare ligated together to generate a capture probe. In some embodiments,an RNA ligase is used to ligate the first and second oligonucleotidetogether. In some embodiments, a DNA ligase is used to ligate the firstand second oligonucleotides sequences together. In some embodiments, theDNA ligase is a T4 DNA ligase. In some embodiments the ligase is Appligase.

In some embodiments, the splint oligonucleotide includes a sequence thatis complementary to the nucleotides of a generated 3′ overhang of thefirst oligonucleotide, or a portion thereof, and a sequence that iscomplementary to the second oligonucleotide, or a portion thereof. Insome embodiments, the splint oligonucleotide includes a sequence that isperfectly complementary (e.g., is 100% complementary) to the nucleotidesof a generated 3′ overhang of the first oligonucleotide, or a portionthereof, and/or a sequence that is perfectly complementary to the secondoligonucleotide, or a portion thereof. In some embodiments, the splintoligonucleotide includes a sequence that is not perfectly complementary(e.g., is not 100% complementary) to the nucleotides of a generated 3′overhang of the first oligonucleotide, or a portion thereof, and/or asequence that is not perfectly complementary to the secondoligonucleotide, or a portion thereof. In some embodiments, the splintoligonucleotide includes a sequence that is at least 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% complementary to the nucleotides of agenerated 3′ overhang of the first oligonucleotide, or a portionthereof, and/or a sequence that is complementary to the secondoligonucleotide, or a portion thereof. In some embodiments, the splintoligonucleotide includes a sequence that is perfectly complementary(e.g., is 100% complementary) to the nucleotides of a generated 3′overhang of the first oligonucleotide, or a portion thereof, but is notperfectly complementary to a sequence that is complementary to thesecond oligonucleotide, or a portion thereof. In some embodiments, thesplint oligonucleotide includes a sequence that is not perfectlycomplementary (e.g., is not 100% complementary) to the nucleotides of agenerated 3′ overhang of the first oligonucleotide, or a portionthereof, but is perfectly complementary to a sequence that iscomplementary to the second oligonucleotide, or a portion thereof. Solong as the splint oligonucleotide is capable of hybridizing to thenucleotides of a generated 3′ overhang of the first oligonucleotide, ora portion thereof, and to a sequence that is complementary to the secondoligonucleotide, or a portion thereof, the splint oligonucleotide neednot have a sequence that is perfectly complementary to either thegenerated 3′ overhang of the first oligonucleotide or to the secondoligonucleotide. The second oligonucleotide can be about 20 to about 80nucleotides long. In some embodiments, the second oligonucleotide can beabout 30 to about 70 nucleotides long. In some embodiments, the secondoligonucleotide can be about 40 to about 60 nucleotides long. In someembodiments, the second oligonucleotide can be about 45 to about 55nucleotides long. The second oligonucleotide can include variousdomains. For example, the second oligonucleotide can include one or moreof a sequence complementary to the splint oligonucleotide, a functionalsequence, a unique molecular identifier, and a capture domain (e.g., anyof the variety of functional sequences, a unique molecular identifiers,and/or a capture domains described herein). In some embodiments of anyof the methods described herein, the capture probes can include a uniquemolecular identifier. In some embodiments of any of the methodsdescribed herein, a unique molecular identifier can be about or at least5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, or 50 nucleotides.

In some embodiments, the capture domain can include a poly(dT) sequence.In some embodiments, the poly(dT) sequence can interact (e.g.,hybridize) with poly(A) sequences of analytes (e.g., mRNA). In someembodiments, the capture domain can include a gene specific sequence.

Capture Probes From Hydrogel Arrays

In some aspects, provided herein are methods for preparing ahigh-density spatially barcoded array, e.g., a high-diversity andhigh-density spatially barcoded array. For example, methods forpreparing a high-density spatially barcoded array can include contactingan array including one or more pluralities of first oligonucleotideswith a hydrogel including one or more pluralities of secondoligonucleotides. In some embodiments, the first oligonucleotide, thesecond oligonucleotide, or both, can have include a capture domain. Thefirst oligonucleotide and the second oligonucleotide can be attached togenerate capture probes. The hydrogel containing the capture probes canbe reduced in size (e.g., shrunk). In some embodiments, methods forpreparing a high-density spatially barcoded array can include contactingan array including one or more pluralities of first oligonucleotideswith a hydrogel including one or more pluralities of secondoligonucleotides; attaching a first oligonucleotide to a secondoligonucleotide to generate a capture probe including a capture domain;reducing the size of the hydrogel including the capture probes bydehydration (e.g., removing water), forming a shrunken hydrogel arrayincluding the one or more pluralities of capture probes and thusgenerating a high-density spatially barcoded array (e.g., spatialarray). In some embodiments, a capture probe is any of the variety ofcapture probes described herein. In some embodiments, the one or morepluralities of capture probes are immobilized in the hydrogel.

The term “partition,” as used herein, can be a verb or a noun. When usedas a verb (e.g., “to partition,” or “partitioning”), the term generallyrefers to the fractionation (subdivision) of a species or sample (e.g.,a polynucleotide) such that one fraction (or subdivision) is sequesteredfrom another. Each sequestered section is referred to using the noun“partition.” In some embodiments, a partition maintains separation ofits own contents from the contents of other partitions. A partition maybe any suitable area in which one fraction of a sample is separated orsequestered from another. In some embodiments, a hydrogel can includeone or more partitions of a species or sample (e.g., a polynucleotide),wherein the hydrogel matrix maintains the separation of the contents ofone partition from the contents of other partitions. In someembodiments, a partition has defined dimensions.

In some embodiments, the one or more pluralities of immobilized firstoligonucleotides are partitioned. In some embodiments, the one or morepluralities of immobilized first oligonucleotides are partitioned into aplurality of first oligonucleotide partitions. In some embodiments, apartitioned plurality of immobilized first oligonucleotides is differentfrom another partitioned plurality of immobilized firstoligonucleotides. In some embodiments, a partitioned plurality ofimmobilized first oligonucleotides are partitioned into features on asubstrate.

In some embodiments, one or more first pluralities of firstoligonucleotides include a barcode sequence (e.g., spatial barcode). Insome embodiments, one or more pluralities of first oligonucleotidesinclude a spatial barcode sequence different from the spatial barcodesequence of one or more other pluralities of first oligonucleotides. Insome embodiments, one or more first oligonucleotide partitions (e.g., apopulation) include a plurality of immobilized first oligonucleotidesincluding a spatial barcode sequence different from the spatial barcodesequence of the plurality of immobilized first oligonucleotides of oneor more other first oligonucleotide partitions. In some embodiments, afirst oligonucleotide of a population of first oligonucleotides in oneor more first oligonucleotide partitions includes a spatial barcode. Insome embodiments, the first oligonucleotide of a population of firstoligonucleotides in one or more first oligonucleotide partitionsincludes a spatial barcode that is different from the spatial barcode ofa first oligonucleotide of a population of first oligonucleotides inanother first oligonucleotide partition. In some embodiments, a spatialbarcode of the first oligonucleotide of a population of firstoligonucleotides in one or more first oligonucleotide partitions isdifferent from a spatial barcode of a second oligonucleotide of apopulation of second oligonucleotides in one or more secondoligonucleotide partitions. In some embodiments, a first oligonucleotidepartition comprises a first population of first oligonucleotidescomprising a spatial barcode that is different from a spatial barcode ofa second population of first oligonucleotides in another firstoligonucleotide partition.

In some embodiments, the one or more pluralities of secondoligonucleotides are immobilized in the hydrogel. In some embodiments,the immobilized second oligonucleotides are partitioned. In someembodiments, the immobilized second oligonucleotides are partitionedinto a first population of immobilized second oligonucleotides (e.g., ina partition). In some embodiments, one or more immobilized secondoligonucleotide partitions include another population of immobilizedsecond oligonucleotides different from the first population ofimmobilized second oligonucleotide partitions. In some embodiments, thefirst immobilized second oligonucleotide of a partitioned population(e.g., a first population) of immobilized second oligonucleotides isdifferent from a second immobilized second oligonucleotide of anotherpartitioned population of immobilized second oligonucleotides. In someembodiments, the first immobilized second oligonucleotide, the secondimmobilized second oligonucleotide, or both include a spatial barcode.In some embodiments, the first immobilized second oligonucleotide of apartitioned population of immobilized second oligonucleotides includes aspatial barcode that is different from a spatial barcode of the secondimmobilized second oligonucleotide of another partitioned population ofimmobilized second oligonucleotides.

In some embodiments, one or more pluralities of second oligonucleotidesinclude a spatial barcode sequence. In some embodiments, one or morepluralities of second oligonucleotides include a spatial barcodesequence different from the spatial barcode sequence of one or morepluralities of second oligonucleotides. In some embodiments, the spatialbarcode sequence of one or more pluralities of first oligonucleotides isdifferent from the spatial barcode sequence of one or more pluralitiesof second oligonucleotides. In some embodiments, one or more secondoligonucleotide partitions include a plurality of secondoligonucleotides including a spatial barcode sequence different from thespatial barcode sequence of the plurality of second oligonucleotides ofone or more second oligonucleotide partitions.

In some embodiments, each first oligonucleotide from the plurality offirst oligonucleotides includes a spatial barcode. In some embodiments,each first oligonucleotide from the plurality of first oligonucleotidesincludes a unique molecular identifier. In some embodiments, each firstoligonucleotide from the plurality of first oligonucleotides includes acleavage domain, a spatial barcode, a unique molecular identifier, and acapture domain. In some embodiments, at least one first oligonucleotidefrom the plurality of first oligonucleotides includes a spatial barcode.In some embodiments, at least one first oligonucleotide from theplurality of first oligonucleotides includes a unique molecularidentifier. In some embodiments, at least one first oligonucleotide fromthe plurality of first oligonucleotides includes a cleavage domain, aspatial barcode, a unique molecular identifier, and a capture domain.

A “capture probe” as used herein can refer to a capture probe that is aproduct of attaching a first oligonucleotide to second oligonucleotideis a hydrogel. For example, one or more domains of the firstoligonucleotide can be attached to one or more domains of a secondoligonucleotide (e.g., a second oligonucleotide in a hydrogel).“Attaching” the first and second oligonucleotide in a hydrogel can beaccomplished by any suitable means described herein. For example, afirst oligonucleotide can be ligated to an end of a secondoligonucleotide. As another example, PCR can be used to attach one ormore domains of a first oligonucleotide to one or more domains of asecond oligonucleotide (e.g., a second oligonucleotide in a hydrogel).

In some embodiments, methods provided herein that include attaching afirst oligonucleotide to second oligonucleotide is a hydrogel can beused to generate a spatial array of relatively high complexity. In someembodiments, a plurality of first oligonucleotides (e.g., partitionedfirst oligonucleotides) is attached to a plurality of secondoligonucleotides (e.g., partitioned second oligonucleotides) to generatea plurality of capture probes such that the plurality of capture probesexhibits a high complexity (e.g., a high sequence complexity) relativeto the complexity of the plurality of first and second oligonucleotides.For example, a plurality of first oligonucleotides can have a pluralityof first partial spatial barcodes, and a plurality of secondoligonucleotides can have a plurality of second partial spatialbarcodes. By attaching the plurality of first and secondoligonucleotides to each other in a combinatorial fashion, a pluralityof spatial barcodes can be generated, which plurality of generatedspatial barcodes can have a higher sequence complexity than the sequencecomplexity of either of the first or second partial spatial barcodes. Asa non-limiting example, if a plurality of first oligonucleotidesincludes a plurality of M first partial spatial barcodes (e.g., theplurality of first oligonucleotides can be partitioned such that each ofthe M first partial spatial barcodes is in a different partition) and aplurality of second oligonucleotides includes a plurality of N secondpartial spatial barcodes (e.g., the plurality of second oligonucleotidescan be partitioned such that each of the N second partial spatialbarcodes is in a different partition), the plurality of first and secondoligonucleotides can be attached in a combinatorial fashion to generatea plurality of spatial barcodes that includes M x N spatial barcodes.

In some embodiments, attaching members of the one or more pluralities offirst oligonucleotides to members of the one or more pluralities ofsecond oligonucleotides to form one or more pluralities of captureprobes includes using a polymerase chain reaction (PCR). In someembodiments, the hydrogel includes PCR reagents such as a DNApolymerase, a plurality of primers, and a plurality of nucleotides. Insome embodiments, members of the plurality of features are amplified(see, e.g., Mitra and Church, Nucleic Acids Res. 15; 27(24):e34 (1999)which is herein incorporated by reference in its entirety).

In some embodiments, chemical ligation can be used to ligate two or moreoligonucleotides (e.g., a first oligonucleotide and a secondoligonucleotide from a hydrogel as described herein). In someembodiments, chemical ligation involves the use of condensing reagents.In some embodiments, condensing reagents are utilized to activate aphosphate group. In some embodiments, condensing reagents may be one ormore of 1- 25 ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI),cyanogen bromide, imidazole derivatives, and 1-hydroxybenzotriazole(HOAt). In some embodiments, functional group pairs selected from one ormore of a nucleophilic group and an electrophilic group, or an alkyneand an azide group are used for chemical ligation. In some embodiments,chemical ligation of two or more oligonucleotides requires a templatestrand that is complementary to the oligonucleotides to be ligated(e.g., a splint oligonucleotide). In some embodiments, the chemicalligation process is similar to oligonucleotide synthesis.

In some embodiments, not all of the domains of a first oligonucleotideare attached to a second oligonucleotide to form a capture probe. Forexample, a capture probe can include 1, 2, 3, 4, or 5 domains of theoriginal first oligonucleotide. In some embodiments, all the domains ofthe first oligonucleotide are attached to the second oligonucleotide.For example, the capture probe includes all the domains of the firstoligonucleotide.

In some embodiments, members of the one or more pluralities of the firstoligonucleotide are cross-linked to a hydrogel.

As used herein “shrinking” or “reducing the size of” a hydrogel refersto any process causing the hydrogel to physically contract and/or thesize of the hydrogel to decrease in volume. For example, the scaffold ofthe gel may shrink or “implode” upon solvent removal (see, e.g., Longand Williams. Science. 2018; 362(6420):1244-1245, and Oran et al.Science. 2018; 362(6420):1281-1285; each of which is incorporated hereinby reference in its entirety). As another example, the process to shrinkor reduce the size of a hydrogel may be one that removes water (e.g., adehydrating process) from the hydrogel. There are many methods known toone of skill in the art for shrinking or reducing the size of ahydrogel. Non-limiting examples of a method to shrink or reduce the sizeof a hydrogel include exposing the hydrogel to one or more of: adehydrating solvent, a salt, heat, a vacuum, lyophilization,desiccation, filtration, and air-drying.

In some embodiments, the removal of water includes a dehydratingsolvent. Non-limiting examples of a dehydrating solvent include a ketone(such as methyl ethyl ketone (MEK)), isopropanol (IPA), acetone,1-butanol, methanol (MeOH), dimethyl sulfoxide (DMSO), glycerol,propylene glycol, ethylene glycol, ethanol, (k) 1,4-dioxane, propylenecarbonate, furfuryl alcohol, N,N-dimethylformamide (DMF), acetonitrile,and an aldehyde (such as formaldehyde or glutaraldehyde). In someembodiments, the solvent includes at least one of an alcohol, a ketone,an aldehyde, acetonitrile, and a combination thereof. In someembodiments, the solvent includes a solvent selected from the groupconsisting of: glycerol, ethanol, acetonitrile, acetone, 2-propanol, anda combination thereof.

In some embodiments, the removal of water includes the use of a salt. Insome embodiments, the salt includes an inorganic salt. Non-limitingexamples of salts that may be used to shrink a hydrogel includeinorganic salts such as aluminum, ammonium, barium, beryllium, calcium,cesium, lithium, magnesium, potassium, rubidium, sodium, and strontiumsalts. Further non-limiting examples of inorganic salts include sodiumchloride, potassium chloride, lithium chloride, cesium chloride, sodiumfluoride, sodium bromide, sodium iodide, sodium nitrite, potassiumsulfate, potassium nitrate, potassium carbonate, potassium bicarbonate,sodium sulfate, sodium nitrate, sodium carbonate, sodium bicarbonate,calcium sulfate, copper oxychloride, calcium chloride, calciumcarbonate, calcium bicarbonate, magnesium sulfate, magnesium nitrate,magnesium chloride, magnesium carbonate, magnesium bicarbonate, ammoniumsulfate, ammonium chloride, ammonium nitrate, ammonium carbonate,ammonium bicarbonate, trisodium phosphate, tripotassium phosphate,calcium phosphate, copper(II) sulfate, sodium sulfide, potassiumsulfide, calcium sulfide, potassium permanganate, iron(II) chloride,iron(III) chloride, iron (2+) sulfate, iron(III) sulfate, iron(II)nitrate, iron(III) nitrate, manganese(II) chloride, manganese(III)chloride, manganese(II) sulfate, manganese(II) nitrate, zinc chloride,zinc nitrate, zinc sulfate, ammonium orthomolybdate, monopotassiumphosphate, nickel(II) sulfate, nickel(II) nitrate, sodium metavanadate,sodium paravanadate, potassium dichromate, ammonium dichromate,antipyonin, ammonium nitrite, potassium fluoride, sodium fluoride,ammonium fluoride, calcium fluoride, chrome alum, potassium alum,potassium iodide, sodium hypochlorite, tin(II) sulfate, tin(II) nitrate,gold selenite, dicesium chromate, potassium perchlorate, calciumperchlorate, aluminium sulphate, lead(II) bisulfate, barium phosphate,barium hydrogen orthophosphate, barium dihydrogen phosphate, silverdichromate, potassium bromate, sodium bromate, sodium iodate, sodiumsilicate, diammonium phosphate, ammonium phosphate, ammonium dihydrogenphosphate, chromium orthophosphate, copper(II) chloride, copper(I)chloride, sodium tetrametaphosphate, potassium heptafluoroniobate, zincphosphate, sodium sulfite, copper(I) nitrate, copper(II) nitrate,potassium silicate, copper(II) carbonate basic, copper(II) carbonatesalts of acrylic acid and sulfopropyl acrylate.

In some embodiments, the removal of water includes an acid. In someembodiments, the hydrogel is exposed to a different pH environment. Forexample, the hydrogel can be exposed to an acidic pH or a basic pH. Insome embodiments, the hydrogel is exposed to a pH of less than about6.5, e.g., a pH of about 6, about 5.5, about 5, about 4.5, about 4,about 3.5, about 3, about 2.5, about 2, about 1.5, or about 1. In someembodiments, the hydrogel is exposed to a pH of greater than about 7.5,e.g., a pH of about 8, about 8.5, about 9, about 9.5, about 10, about10.5, about 11, about 11.5, 10 about 12, about 12.5, about 13, about13.5, or about 14.

In some embodiments, the removal of water includes a dehydrating processsuch as heat, a vacuum, lyophilization, desiccation, filtration, andair-drying. In some embodiments, the hydrogel is exposed to an increasedtemperature, e.g., a higher temperature than about room temperature. Forexample, a temperature of about 37° C., 38.15° C., 39° C., 40° C., 41°C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50°C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59°C. 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68°C., 69° C., or 70° C., or higher. As another example, the hydrogel isexposed to an increase in temperature from about 37° C., 38° C., 39° C.,40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C.,or 49° C. to about 50° C., 51.20° C., 52° C., 53° C., 54° C., 55° C.,56° C., 57° C., 58° C., 59° C. 60° C., 61° C., 62° C., 63° C., 64° C.,65° C., 66° C., 67° C., 68° C., 69° C., or 70° C., or higher).

In some embodiments, more than one method to shrink or reduce the sizeof a hydrogel can be used. For example, a hydrogel can be exposed to anacid and subsequently dehydrated by another method such as heat, avacuum, lyophilization, desiccation, filtration, or a combinationthereof.

In some embodiments, the hydrogel can further include a cross-linkingagent. The cross linking agent may help stabilize the hydrogel and/ormodulate the extent the hydrogel shrinks. Non-limiting examples of across-linking agent include disuccinimidyl suberate (DSS),dimethylsuberimidate (DMS), formalin, dimethyladipimidate (DMA),dithio-bis(succinimidyl propionate) (DSP), disuccinimidyl tartrate(DST), and ethylene glycol bis(succinimidyl succinate) (EGS). In someembodiments, the shrunken hydrogel is at least about 2-fold smaller in alinear dimension (e.g., along one axis) than the pre-shrunk hydrogel.For example, at least about 2.5, about 3, about 4, about 5, about 6,about 7, about 8, about 9, about 10, or more fold smaller in a lineardimension than the pre-shrunk hydrogel. In some embodiments, a shrunkenhydrogel is about 2 to about 10 fold smaller in a linear dimension thanthe pre-shrunk hydrogel. For example, about 2 to about 3, about 2 toabout 4, about 2 to about 5, about 2 to about 6, about 2 to about 7,about 2 to about 8, about 2 to about 9, about 9 to about 10, about 8 toabout 10, about 7 to about 10, about 6 to about 10, about 5 to about 10,about 4 to about 10, or about 3 to about 10. In some embodiments, ashrunken hydrogel is about 2 to about 4, about 3 to about 5, about 4 toabout 6, about 5 to about 7, about 6 to about 8 fold smaller in a lineardimension than the pre-shrunk hydrogel. In some embodiments, a shrunkenhydrogel is about 2, about 3, about 4, about 5, about 6, about 7, about8, about 9, or about 10 fold smaller in a linear dimension than thepre-shrunk hydrogel. In some embodiments, the shrunken hydrogel is about2 to about 10 fold smaller in a linear dimension than the size of thehydrogel prior to reducing the size of the pre-shrunk hydrogel.

In some embodiments, the size of the hydrogel is reduced along more thanone axes, e.g., along 2 or 3 axes. In some embodiments, each axisintersects each other axis at 90 degrees. In some embodiments, the sizeof the hydrogel along the first axis is about 2 to about 10 or more foldsmaller than the pre-shrunk hydrogel, e.g., about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10 or more foldsmaller than the pre-shrunk hydrogel. In some embodiments, the size ofthe hydrogel along the second axis is about 2 to about 10 or more foldsmaller than the pre-shrunk hydrogel, e.g., about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10 or more foldsmaller than the pre-shrunk hydrogel. In some embodiments, the size ofthe hydrogel along the third axis is about 2 to about 10 or more foldsmaller than the preshrunk hydrogel, e.g., about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10 or more foldsmaller than the pre-shrunk hydrogel. In some embodiments, the reductionin the size of the hydrogel is isometric.

In some embodiments, a hydrogel is prepared from hydrogel subunitsincluding at least one of: acrylamide, bis-acrylamide, a poly(ethyleneglycol), a polyaliphatic polyurethane, a polyether polyurethane, apolyester polyurethane, a polyethylene copolymer, a polyamide, apolyvinyl alcohol, polypropylene glycol, polytetramethylene oxide,polyvinyl pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate),poly(hydroxyethyl methacrylate), collagen, hyaluronic acid, chitosan,dextran, agarose, gelatin, alginate, a protein polymer, methylcellulose,and any combination thereof.

In some embodiments, the size of a hydrogel including capture probes isreduced, thus increasing the density of the capture probes. In someembodiments, the volume of a partition of members of the plurality ofcapture probes in a hydrogel is reduced when the size of the hydrogel isreduced.

In some embodiments, a high-density spatially barcoded array (e.g., ahigh density spatially barcoded array as described herein) can be usedfor spatial analysis of analytes present in the biological sample. Forexample, methods for spatially determining the location of an analyte ora plurality of analytes in a biological sample can include providing ahigh-density spatially barcoded array as described herein; contactingthe biological sample with the high-density spatially barcoded arrayunder conditions such that one or more analytes present in thebiological sample are captured by one or more capture probes of thearray; and determining the spatial location of the captured analytes byany of the methods described herein.

Methods for Spatial Analysis of Analytes

In some embodiments, a high-density spatially barcoded array (e.g., ahigh density spatially barcoded array as described herein) can be usedfor spatial analysis of analytes present in a biological sample. Forexample, methods for spatially determining a location of an analyte or aplurality of analytes in a biological sample can include providing ahigh-density spatially barcoded array as described herein; contactingthe biological sample with the high-density spatially barcoded arrayunder conditions such that one or more analytes present in thebiological sample are captured by one or more capture probes of thearray; and determining the spatial location of the captured analyte(s).

In some aspects, provided herein methods for building an array (e.g., aspatial array, e.g. a high-density spatial array). In some embodiments,methods of generating an array comprise: (a) providing an substrateincluding a plurality of features immobilized on the substrate, whereinthe features (e.g., each feature or a substantial portion thereof)comprises a plurality of first oligonucleotides, and wherein the firstoligonucleotides (e.g., each first nucleotide or a substantial portionthereof) include a first spatial barcode (e.g., a first partial spatialbarcode); (b) selecting one or more first oligonucleotides from theplurality of first oligonucleotides; (c) delivering a secondoligonucleotide to the one or more selected first oligonucleotides,wherein the second oligonucleotide includes a second spatial barcode(e.g., a second partial spatial barcode) and a capture domain; and (d)attaching the second oligonucleotide to the one or more selected firstoligonucleotides, thereby generating one or more capture probesincluding the first spatial barcode and the second spatial barcode,thereby generating an array.

In some embodiments, each feature of the plurality of features isassociated with one of a number of different first spatial barcodes. Insome embodiments, each second oligonucleotide is associated with one ofa number of different second barcodes.

In some embodiments, step (d) includes ligating the secondoligonucleotide to the one or more selected first oligonucleotides. Insome embodiments, the method includes ligating the secondoligonucleotide to the one or more selected first oligonucleotides usinga ligase. In some embodiments, the second oligonucleotide is attached tothe one or more selected first oligonucleotides by a chemical couplingreaction. In some embodiments, one or more of (e.g., each of) the firstoligonucleotides includes a 3′ end that is masked by a protective group(e.g., a photocleavable protective group). In some embodiments, themethod includes removing the photocleavable protective group from theone or more selected first oligonucleotides prior to delivering a secondoligonucleotide to the one or more selected first oligonucleotides. Insome embodiments, the protective group is a photocleavable protectivegroup (e.g., any of the variety of a photocleavable protective groupsdescribed herein or otherwise known in the art). In some embodiments,the protective group is removed by exposing the one or more selectedfirst oligonucleotides to light. In some embodiments, the protectivegroup coupled to the one or more selected first oligonucleotides is aphotomask (e.g., any of the variety of a photomasks described herein orotherwise known in the art). In some embodiments, the photomask isremoved by exposing the one or more selected first oligonucleotides tolight.

In some embodiments, the selected first oligonucleotide(s) are separatedfrom the other first oligonucleotides of the plurality by one or morephysical barriers, wherein the one or more physical barriers form one ormore chambers, and wherein the second oligonucleotide is delivered tothe one or more chambers including the one or more selected firstoligonucleotides. In some embodiments, the one or more physical barriersinclude a hydrophobic surface. In some embodiments, the one or morephysical barriers include one or more chamber walls having a taperedend. In some embodiments, the selected first oligonucleotide(s) areseparated from the other first oligonucleotides of the plurality, butare not separated by one or more physical barriers. For example, theselected first oligonucleotide(s) can be separated from the other firstoligonucleotides of the plurality simply by virtue of their spatiallocations. In some embodiments, the second oligonucleotide includes aunique molecular identifier. In some embodiments, the secondoligonucleotide includes a 3′ end that is masked. In some embodiments, afree 3′ —OH group on the capture probes is created.

In some embodiments, methods of generating an array comprise: (a)providing an array including two or more partitions, wherein thepartitions (e.g., each partition or a substantial portion thereof)includes a plurality of features immobilized on a substrate, wherein thefeatures (e.g., each feature or a substantial portion thereof) includesa plurality of first oligonucleotides, and wherein the firstoligonucleotides (e.g., each first oligonucleotide or a substantialportion thereof) include a first spatial barcode(e.g., a first partialspatial barcode); (b) delivering a second oligonucleotide to a firstpartition, wherein the second oligonucleotide includes a second barcode(e.g., a second partial spatial barcode)and a capture domain; and (c)attaching the second oligonucleotide to one or more firstoligonucleotides in the first partition, thereby generating one or morecapture probes including the first spatial barcode and the secondspatial barcode in the first partition, thereby generating an array.

In some embodiments, methods provided herein further include: (d)delivering a third oligonucleotide to a second partition of the two ormore partitions, wherein the third oligonucleotide includes a thirdspatial barcode (e.g., a third partial spatial barcode); and (e)attaching the third oligonucleotide to a fourth oligonucleotide in thesecond partition, wherein the fourth oligonucleotide includes the firstspatial barcode (e.g., the first partial spatial barcode), therebygenerating capture probes including the first spatial barcode and thethird spatial barcode in the second partition. In some embodiments,methods provided herein further include: (d) delivering a thirdoligonucleotide to a second partition, wherein the third oligonucleotideincludes a third spatial barcode (e.g., a third partial spatialbarcode); and (e) attaching the third oligonucleotide to a fourtholigonucleotide in the second partition, wherein the fourtholigonucleotide includes the fourth spatial barcode (e.g., a fourthpartial spatial barcode) thereby generating capture probes including thefourth spatial barcode and the third spatial barcode in the secondpartition.

In some embodiments, the two or more partitions are separated by one ormore physical barriers. In some embodiments, the one or more physicalbarriers are printed on the substrate. In some embodiments, the one ormore physical barriers form one or more chambers including one or morechamber walls. In some embodiments, the one or more chamber wallsinclude a hydrophobic surface. In some embodiments, the one or morechamber walls include a tapered end in contact with the substrate. Insome embodiments, the second oligonucleotide is delivered using amicrofluidic device.

Chemical Ligation of Oligonucleotides

In some embodiments, chemical ligation can be used to ligate two or moreoligonucleotides. In some embodiments, chemical ligation involves theuse of condensing reagents. In some embodiments, condensing reagents areutilized to activate a phosphate group. In some embodiments, condensingreagents may be one or more of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), cyanogen bromide, imidazole derivatives, and1-hydroxybenzotriazole (HOAt). In some embodiments, functional grouppairs selected from one or more of a nucleophilic group and anelectrophilic group, or an alkyne and an azide group are used forchemical ligation. In some embodiments, chemical ligation of two or moreoligonucleotides requires a template strand that is complementary to theoligonucleotides to be ligated (e.g., a splint oligonucleotide). In someembodiments, the chemical ligation process is similar to oligonucleotidesynthesis.

Photocleavable Protective Groups

In some embodiments, a photocleavable protective group (e.g. anyphotocleavable, photoremovable, photoreleasable, photoactivatable, orphotosensitive protective group described herein or known in the art)can be coupled to one or both ends of an oligonucleotide on a substrate,a surface probe (e.g. any of the surface probes described herein), or acapture probe (e.g., any of the capture probes described herein). Insome embodiments, a photocleavable protective group is a chemicalmodification to a molecule that can be removed with light. In someembodiments, the photocleavable protective group (PPG) is at least oneof a nitrobenzyl-based PPG, a carbonyl-based PPG, or a benzyl-based PPG.In some embodiments, the photocleavable protective group is removed fromone or more surface probes by exposing the one or more surface probes toa light.

Shallow Sequencing

As used herein, the coverage or depth of sequencing is the number ofunique reads that include a given nucleotide in a reconstructedsequence. As used herein, shallow sequencing is a small or low number ofunique reads of an identified analyte. In some embodiments, shallowsequencing can be performed to identify a spatial region of interest. Insome embodiments, shallow sequencing can be performed on an entirebiological sample. In some embodiments, shallow sequencing can beperformed on an identified region of a biological sample (e.g., a regionof interest). In some embodiments, shallow sequencing can be performedafter identifying and/or selecting a region of a biological sample bysome other method (e.g., immunostaining).

Target Spatial Barcodes

In some embodiments, a target spatial barcode (e.g., sometimes referredto herein as a target barcode sequence) is a barcode sequencecorresponding to a spatial region of interest. In some embodiments, atarget spatial barcode is or includes a spatial barcode. In someembodiments, a target spatial barcode is identified by identifying aspatial region of interest and identifying the spatial barcodeassociated with the spatial region of interest on the array. In someembodiments, a target spatial barcode is identified by performingshallow sequencing, and selecting a spatial barcode based on thecaptured analytes. As used herein, the coverage or depth of sequencingis the number of unique reads that include a given nucleotide in areconstructed sequence. As used herein, shallow sequencing is a small orlow number of unique reads of an identified analyte. In someembodiments, shallow sequencing can be performed to identify a spatialregion of interest. In some embodiments, shallow sequencing can beperformed on an entire biological sample. In some embodiments, shallowsequencing can be performed on an identified region of a biologicalsample (e.g., a region of interest). In some embodiments, shallowsequencing can be performed after identifying and/or selecting a regionof a biological sample by some other method (e.g., immunostaining). Insome embodiments, a target spatial barcode is identified by aco-localization of a target spatial barcode with one or more additionalspatial barcode sequences. In some embodiments, a target spatial barcodeis identified by imaging. In some embodiments, imaging includesimmunohistochemistry and/or other staining or microscopy methods. Insome embodiments, a target spatial barcode is identified manually. Insome embodiments, a target spatial barcode is identified using acomputer. In some embodiments, a target spatial barcode is identifiedalgorithmically. In some embodiments, the target spatial barcodecorresponds to a plurality of spatial barcode sequences that identify aregion, a sub-region, a sub-sub-region, or further spatial demarcation.In some embodiments, a target spatial barcode is used to identify andcharacterize captured analytes at single-cell resolution. In someembodiments, the identifying can include sequencing a plurality ofcapture probes to generate a plurality of sequencing reads, andanalyzing the plurality of sequencing reads to identify the at least onetarget spatial barcode. In some embodiments, each of the plurality ofcapture probes can further include one or more functional sequencesselected from the group consisting of a sequencing primer sequence and asequence configured to attach to a flow cell of a sequencer. In someembodiments, the plurality of capture probes can include, from 5′ to 3′,a first functional domain (e.g., adapter sequence), a sequence derivedfrom a template nucleic acid, and a second adapter sequence. The firstadapter sequence can include a first sequence configured to attach to aflow cell of a sequencer, a first sequencing primer sequence, and aspatial barcode. The second adapter sequence can include a secondsequencing primer sequence and a second sequence configured to attach toa flow cell of a sequencer. The second adapter sequence can furtherinclude an index sequence.

Enriching Barcoded Nucleic Acids

In an aspect, the present disclosure provides methods of enriching asample for target spatial barcoded nucleic acids. The methods caninclude providing a plurality of capture probes including a plurality ofdifferent spatial barcodes and a target capture probe of the populationof captures, identifying at least one target spatial barcode from theplurality of different target spatial barcodes, and enriching thepopulation of target capture probes including the spatial barcode fromthe plurality of capture probes.

In an aspect, the present disclosure provides methods of enriching asample for target spatial barcoded nucleic acids. The methods caninclude providing a plurality of capture probes including a plurality ofdifferent spatial barcodes, identifying at least one target spatialbarcode from the plurality of different target spatial barcodes, andenriching the analyte detected by (e.g., specifically bound to thecapture probe) the capture probes including the at least one targetspatial barcode (e.g., sometimes referred to herein as a target barcodesequence). Enriching can be performed by purifying capture probesincluding the at least one target spatial barcode from a plurality ofcapture probes. Additionally or alternatively, enriching can beperformed by amplifying (e.g., increasing the numbers of) capture probesincluding the at least one target spatial barcode from a plurality ofcapture probes. The amplification can include any of the variety ofamplification methods described herein, including isothermalamplification and/or PCR. Enriching can be performed by both purifyingand amplifying the capture probe including the at least one targetspatial barcode from a plurality of capture probes.

Spatial barcodes can be included in capture probes. For example, thecapture probes can be co-partitioned into a partition with spatialbarcodes to facilitate the barcoding reaction for spatial analysis. Thespatial barcodes can be attached to a bead (e.g., in the context of acapture probe). The bead can be a gel bead. In some embodiments, thespatial barcodes are releasably attached to the bead (e.g., gel bead) asdescribed elsewhere herein (e.g., via a cleavage domain). The pluralityof capture probes can be spatially barcoded as described elsewhereherein.

In some embodiments, methods provided herein can include enrichingnucleic acids including the at least one target spatial barcode (e.g.,capture probes that have been extended using a captured analytes as atemplate). The enriching can include performing a nucleic acidamplification reaction on at least one capture probe including the atleast one target spatial barcode to generate an enriched plurality ofcapture probes including the at least one target spatial barcode. Thenucleic acid amplification reaction can be a polymerase chain reaction(PCR). The nucleic acid extension amplification reaction can be anisothermal amplification reaction. The nucleic acid amplificationreaction can include a polymerase. The nucleic acid extension reactioncan result in amplification of the nucleic acid.

In some embodiments, methods provided herein can further includeperforming an additional amplification reaction (e.g., PCR) on theenriched plurality of capture probes including the at least one targetspatial barcode. The amplification reaction PCR can include use of anadditional primer including a sequence specific for the enrichedplurality of capture probes including the at least one target spatialbarcode sequence. In some embodiments, the additional primer can includea sequence specific for the spatial barcode of the enriched plurality ofcapture probes including the at least one target spatial barcode. Insome embodiments, the additional primer can include a sequencesubstantially specific for the spatial barcode sequence of the enrichedplurality of capture probes including the at least one target spatialbarcode. For example, the additional primer can include a sequencehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more sequencecomplementarity or sequence identity to the spatial barcode sequence ofthe enriched plurality of capture probes including the at least onetarget spatial barcode. In some embodiments, the additional primer caninclude a sequence specific for a 5′ region of a spatial barcodesequence of the enriched plurality of capture probes including the atleast one target spatial barcode. In some cases, the additional primercan include a sequence that has no (or low) sequence complementarity oridentity to the spatial barcode sequence of the enriched plurality ofcapture probes including the at least one target spatial barcode.

In some embodiments, the additional primer can include one or morefunctional sequences that facilitate sequencing of the enrichedplurality of capture probes. The one or more functional sequences caninclude a sequencing primer sequence. The one or more functionalsequences can include a sequence configured to attach to a flow cell ofa sequencer.

In some embodiments, methods provided herein can further include,subsequent to the nucleic acid amplification reaction, performing a sizeselection process to remove unincorporated primers. The size selectionprocess can include using a gel or gel-like polymer. The size selectionprocess can use gel electrophoresis. In some cases, the size selectionprocess can include using a substrate. The substrate can be an array.

In some embodiments, the enriched plurality of nucleic acids can includean affinity group. The affinity group can be incorporated to theenriched plurality of the nucleic acid via a polymerization reactionwith a primer including the affinity group. The affinity group can beincorporated via enzymatic reaction with a nucleotide and the affinitygroup. The affinity group may be attached covalently or non-covalently.The method can further include coupling the enriched plurality ofcapture probes including the affinity group to a substrate specific forthe affinity group. The substrate can be an array. The substrate can bea polymer matrix. The substrate can be a glass slide. The substrate canbe a resin. In some embodiments, the substrate can include an antibodyor antibody fragment. In some embodiments, the substrate includes avidinor streptavidin and the affinity group includes (or is) biotin. In someembodiments, the substrate includes maltose and the affinity groupincludes (or is) maltose binding protein. In some embodiments, thesubstrate includes maltose-binding protein and the affinity groupincludes (or is) maltose.

In some embodiments, prior to the nucleic acid amplification reaction, afirst capture probe complementary to a first portion of the at least onetarget spatial barcode and a second capture probe complementary to asecond portion of the at least one target spatial barcode can behybridized. The first capture probe can be ligated to the second captureprobe to generate the primer including the sequence specific for the atleast one target spatial barcode. The ligating can include using a DNAligase.

In some embodiments, the enriching can result in at least 10-foldenrichment of capture probes including the at least one target spatialbarcode. In some embodiments, the enriching can result in at least20-fold enrichment of capture probes including the at least one targetspatial barcode. The enrichment of nucleic acids can be observed by anincrease in the percentage of nucleic acids including the at least onetarget spatial barcode in the enriched plurality of capture probescompared to the percent amount of nucleic acids including the at leastone target spatial barcode in the plurality of capture probes. Theenriching can result in at least 50-fold enrichment of capture probesincluding the at least one target spatial barcode. The enriching canresult in at least 100-fold enrichment of capture probes including theat least one target spatial barcode. The enriching can result in atleast 200-fold enrichment of capture probes including the at least onetarget spatial barcode. The enriching can result in at least 500-foldenrichment of capture probes including the at least one target spatialbarcode. The enriching can result in at least 1000-fold enrichment ofcapture probes including the at least one target spatial barcode.

Primers used for extension or amplification reactions described hereincan be a specific length or include a specific number of nucleotides.For example, the primers may be at least about 5, about 6, about 7,about 8, about 9, about 10, about 12, about 14, about 16, about 18,about 20, about 22, about 24, about 25, about 26, about 28, about 30,about 40, about 50 or more nucleotides long. Alternatively or inaddition, the primers can be no more than about 50, about 40, about 30,about 28, about 26, about 25, about 24, about 22, about 20, about 18,about 16, about 14, about 12, about 10, about 9, about 8, about 7, about6, about 5, or fewer nucleotides long.

The primers may have a specific melting temperature. The meltingtemperature of the primer can be at least about 35° C. (°C), 40° C., 41°C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50°C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59°C. 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., or higher. Themelting temperature of the primer can be no more than about 35° C., 40°C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49°C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58°C., 59° C. 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., or lower.

In some cases, primers are ligated together to form a longer primer. Thelonger primer can be subsequently used for any PCR reactions, asdescribed elsewhere herein. Ligating together shorter primers into alonger primer can increase the stringency of binding such that thetargeted spatial barcode is enriched. For example, the shorter primerscan be able to distinguish mismatches better and thus bind only tosequences with perfect or near perfect complementarity. The longerprimer can be more able to accommodate mismatches in sequences and bindto sequences that are not the targeted barcode. In some cases, more than2 primers are ligated together to form a longer primer. For example, atleast about 3, 4, 5, 6, 7, 8, 9, 10, or more primers can be ligatedtogether to form a longer primer.

In some embodiments, the primer includes an affinity group and theenriched plurality of capture probes includes the affinity group. Theaffinity group can include biotin. The affinity group can includestreptavidin or avidin. The affinity group can include dioxigenin. Theaffinity group can include a protein or polypeptide. The affinity groupcan include an antibody or antibody fragment. The affinity group caninclude a receptor or receptor ligand. The affinity group can includes asugar, a monosaccharide, a disaccharide, or a polysaccharide. Theaffinity group can include maltose.

Methods of the present disclosure can be used for various applications,such as, processing a single analyte (e.g., RNA, DNA, or protein) ormultiple analytes (e.g., DNA and RNA, DNA and protein, RNA and protein,or RNA, DNA, and protein) from a single cell. For example, multipleanalytes from the biological sample can be processed. The multipleanalytes can be from the single cell. This can enable, for example,simultaneous proteomic, transcriptomic and genomic analysis of the cell(e.g., multiplexing).

Hierarchal Barcode Enrichment

In some aspects, the present disclosure provides methods of spatiallydesigning or designating spatial barcodes, such that the position of thespatial barcodes are specifically identified. In some embodiments, aparticular individual feature on a spatial array (e.g., a spot or bead)can contain more than one spatial barcode, wherein each spatial barcodecorresponds to a particular defined region on the spotted array. Forexample, multiple spatial barcode sequences associated with oneparticular feature on an array can provide a stronger address orattribution to a given location by providing duplicate or independentconfirmation of the location. In some cases, the multiple spatialbarcodes represent increasing specificity of the location of theparticular feature. In a non-limiting example, a particular feature canbe coded with two different spatial barcodes, wherein each spatialbarcode identifies a particular defined region within the array, and afeature possessing both spatial barcodes identifies the sub-region wheretwo defined regions overlap, e.g., such as the overlapping portion of aVenn diagram. In another non-limiting example, a particular feature canbe coded with three or more different spatial barcodes, wherein thefirst spatial barcode identifies a first region within the array, thesecond spatial barcode (e.g., sub region spatial barcode, second commonspatial barcode) identifies a second region, wherein the second regionis a subregion entirely within the first region (a sub region), and thethird spatial barcode (e.g., sub-sub region spatial barcode, thirdcommon spatial barcode) identifies a third region, wherein the thirdregion is a subregion entirely within the first and second subregions (asub region), and so on. In some embodiments, each capture probe has aunique molecular identifier (UMI). In some embodiments, each captureprobe has a UMI, as well as one or more target spatial barcodescorresponding to one or more regions or sub regions on an array.

In some embodiments, an array has multiple features (also sometimesreferred to as “regions”, “spots”, or “locations”) and subregions. Insome embodiments, an array has 10 features or more. In some embodiments,an array has 20, 30, 40, 50, 60, 70, 80, 90, 100 features, or more. Insome embodiments, an array has 100, 200, 300, 400, 500, 600, 700, 800,900, 1000 features or more. In some embodiments, an array has 1000,2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 1000 features or more.In some embodiments, the features are identical in size. In someembodiments, the features are not identical in size. The features maytake any shape, including but not limited to square, rectangle, circle,oval, pentagon, hexagon, octagon arc, arches, lens, crescent, and anycombinations thereof. In some embodiments, a feature of an array hasmultiple subregions. In some embodiments, a feature of an array has 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000 subregions or more. In some embodiments, the subregions areidentical in size to other subregions. In some embodiments, thesubregions are not identical in size to other subregions. In someembodiments, the subregions are completely surrounded by a largerregion. In some embodiments, subregions are partially surrounded by alarger region. In some embodiments, different subregions of a featureinclude different regional spatial barcodes that are characteristic ofthe given subregions. In some embodiments, different subregions of afeature include different regional spatial barcodes that arecharacteristic of the given subregions and a common spatial barcode thatis characteristic of the feature. Such embodiments permit enrichment ofcapture analytes having the common spatial barcode and/or one or moreregional spatial barcodes (e.g., via any of the variety of enrichmentmethods described herein). In some embodiments, an array has multiplesub-subregions. In some embodiments, an array has 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000sub-subregions or more. In some embodiments, the sub-subregions areidentical in size to other sub-subregions. In some embodiments, thesub-subregions are not identical in size to other sub-subregions. Insome embodiments, sub-subregions are completely surrounded by a largerregion. In some embodiments, the larger region is a subregion. In someembodiments, the larger region is a region. In some embodiments,sub-subregions are partially surrounded by a larger subregion. In someembodiments, sub-subregions are partially surrounded by a larger region.In some embodiments, different sub-subregions of a subregion includedifferent sub-regional spatial barcodes that are characteristic of thegiven sub-subregions. In some embodiments, different sub-subregions of afeature include different sub-regional spatial barcodes that arecharacteristic of the given subregions and a common regional spatialbarcode that is characteristic of the region. In some embodiments,different sub-subregions of a feature include different sub-regionalspatial barcodes that are characteristic of the given subregions, acommon regional barcode that is characteristic of the region, and acommon spatial barcode that is characteristic of the feature. Suchembodiments permit enrichment of capture analytes having the commonspatial barcode and/or one or more regional spatial barcodes (e.g., viaany of the variety of enrichment methods described herein). In someembodiments, individual features or spots or beads include a region. Insome embodiments, individual features of spots or beads include asubregion. In some embodiments, individual features or spots or beadsinclude a sub-subregion. In some embodiments, individual features orspots or beads include a region corresponding to single cell resolutionof the biological sample.

In some embodiments, the present disclosure provides methods ofenriching a sample including more than one analyte specifically bound tothe capture probe. The methods can include providing a plurality ofcapture probes including a plurality of different spatial barcodes,identifying more than one target spatial barcode (e.g., sometimesreferred to as a target barcode sequence) from the plurality ofdifferent spatial barcodes, and enriching capture probes including themore than one target spatial barcode. Enriching can be performed bypurifying capture probes including the more than one target spatialbarcode from a plurality of capture probes. Enriching can be performedby amplifying (e.g., increasing the numbers of) capture probes includingthe more than one target spatial barcode from a plurality of captureprobes. Enriching can be performed by both purifying and amplifying thecapture probe including the more than one target spatial barcode from aplurality of capture probes. Enriching can be performed using targetspatial barcodes associated with smaller regions. Enriching can beperformed using target spatial barcodes of increasing specificity to aparticular feature or spot or location. For example, enriching can beperformed by amplifying and/or purifying a first plurality of captureprobes including a target spatial barcode that is associated with afirst spatial location (e.g., a first feature, a first subregion, and/ora first sub-subregion). The enriching may further include amplifyingand/or purifying a second plurality of capture probes including a targetspatial barcode that is associated with a second spatial location (e.g.,a second feature, a second subregion, and/or a second sub-subregion)that is partially or completely within the first spatial location. Theenriching can further include amplifying and/or purifying a secondplurality of capture probes including a target spatial barcode that isassociated with a second spatial location that is partially within thefirst region. The enriching can further include amplifying and/orpurifying a second plurality of capture probes including a targetspatial barcode that is associated with a second spatial location thatis completely within the first spatial location. The enriching canfurther include amplifying and/or purifying a third plurality of captureprobes including a target spatial barcode that is associated with athird spatial location that is partially or completely within the firstand second spatial locations. The enriching can further includeamplifying and/or purifying a third plurality of capture probesincluding a target spatial barcode that is associated with a thirdspatial location that is partially within the first and second spatiallocations. The enriching can further include amplifying and/or purifyinga third plurality of capture probes including a target spatial barcodethat is associated with a third spatial location that is completelywithin the first and second spatial locations. The enriching can furtherinclude amplifying and/or purifying a fourth plurality of capture probesincluding additional target spatial barcodes that are associated withfour or more spatial locations that are partially or completely withinthe first, second, and third spatial locations. The enriching mayfurther include amplifying and/or purifying a fifth plurality of captureprobes including additional target spatial barcodes that are associatedwith five or more spatial locations that are partially or completelywithin the first, second, third, and fourth spatial locations. Theenriching can further include amplifying and/or purifying additionalpluralities of capture probes including additional target spatialbarcodes that are associated with 6, 7, 8, 9, 10, or more spatiallocations that are partially or completely within the previouslyidentified regions and/or sub regions.

In some embodiments, the method can include enriching nucleic acidsincluding more than one target spatial barcode. The enriching caninclude performing a nucleic acid extension reaction using a captureprobe including more than one target spatial barcode and a primerincluding a sequence specific for more than one target spatial barcodeto generate an enriched plurality of capture probes including more thanone target spatial barcode. The nucleic acid extension reaction can be apolymerase chain reaction (PCR). The nucleic acid extension reaction caninclude a polymerase. The nucleic acid extension reaction can result inamplification of the nucleic acid.

In some embodiments, methods provided herein may further includeperforming additional rounds of PCR on the enriched plurality of captureprobes corresponding to the number of spatial barcode sequences of agiven location. The additional rounds of PCR may include use ofadditional primers including a sequence specific for the additionaltarget spatial barcode (e.g., target spatial barcode associated with aregion, a subregion, or sub-subregion of interest). In some embodiments,the additional primers may include a sequence specific for the spatialbarcode sequence of the enriched plurality of capture probes includingmore than one target spatial barcode. In some embodiments, theadditional primers may include a sequence substantially specific for anadditional target spatial barcode of the enriched plurality of captureprobes including more than one target spatial barcode. For example, theadditional primer may include a sequence having at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.5%, 99.9% or more sequence complementarity or sequence identityto a target spatial barcode of the enriched plurality of capture probesincluding the more than one target spatial barcode. In some embodiments,the additional primers may include a sequence specific for a 5′ regionof a spatial barcode sequence of the enriched plurality of captureprobes including more than one target spatial barcode. In some cases,the additional primer can includes a sequence that has no (or low)sequence complementarity or identity to one or more spatial barcodesequences of the enriched plurality of capture probes including morethan one target spatial barcode.

In some embodiments, each spatial barcode sequence of the plurality ofdifferent spatial barcode sequences can identify a capture probe asderived from a single cell. Alternatively or in addition, each spatialbarcode sequence of the plurality of different spatial barcode sequencesmay identify a capture probe as derived from a single partition.

Identifying a Spatial Region of Interest

In some embodiments, identifying a spatial region of interest includesany technique for observing and/or locating an area within a tissue. Insome embodiments, identifying a spatial region of interest includesimmunostaining techniques, including but not limited to,immunohistochemistry techniques. In some embodiments, identifying aspatial region of interest includes microscopy techniques, including butnot limited to, optical microscopy, fluorescence microscopy, andconfocal microscopy. In some embodiments, identifying a spatial regionof interest includes visual inspection. In some embodiments, identifyinga spatial region of interest includes shallow sequencing a spatialregion. In some embodiments, identifying a spatial region of interestincludes shallow sequencing the entire biological sample. Any of thevariety of other methods described herein or otherwise known in the artto identify a spatial region of interest can be used.

Generating Spatial Arrays With Light

In at least one embodiment of the compositions and methods of thepresent disclosure, a plurality of first oligonucleotide are attached toa substrate by any suitable means that immobilizes the firstoligonucleotide by either their 3′ or 5′ end in a region of thesubstrate and allows the unattached end to be available for furthersynthesis. Further synthesis can include in situ base-by-base step-wisesynthesis (e.g., phosphoramidite chemistry) or biosynthesis via enzymereactions (e.g., DNA polymerization or reverse transcription). In someembodiments, the means of attachment is also be suitable to allow atleast a portion of the first oligonucleotide to be capable ofhybridization.

In at least one embodiment of the compositions and methods of thepresent disclosure, the plurality of first oligonucleotides are attachedto a substrate by a covalent linkage. Accordingly, in at least oneembodiment, the plurality of first oligonucleotides are attached to thesubstrate by a chemical reaction that forms one or more covalent bondsbetween a functional group on the substrate (or a linker group attachedto the substrate) and a functional group on the 3′ - or 5′-end of thefirst oligonucleotide (or a linker group attached to the 3′ or 5′-end ofthe first oligonucleotide). Such functional groups can include anaturally occurring nucleotide functional group, e.g. a terminalhydroxyl group, or a non-natural functional group introduced duringoligonucleotide synthesis, such as a 5′-amine group. Reagents andtechniques for chemically modifying substrate materials and/ornucleotides with functional groups that are useful in the methods of thepresent disclosure are well known in the art. For example, functionalgroups that may be introduced on a substrate that allow for attachmentof a first oligonucleotide (e.g., 5′ -amine modified oligonucleotide) toa substrate include linkers with available carboxy, aldehyde or epoxygroups. Other functionalized linker compounds are known to the personskilled in the art.

The first oligonucleotide of the plurality of first oligonucleotides tobe attached to a substrate in preparing a spatial array of the presentdisclosure can be understood as having at least three exemplaryoligonucleotide: a first oligonucleotide of about 2-mer to about 40-merthat is attached to the substrate through either its 3′-end or its5′-end; a second oligonucleotide that is attached to the unattached endof the first, and includes a spatial barcode sequence of about 8-mer toabout 30-mer in length that indicates the region on the substrate wherethe second is located (e.g., a spatial barcode); and a plurality ofthird oligonucleotide s of about 5-mer to about 50-mer attached to thesecond oligonucleotide, wherein the third oligonucleotide includes acapture sequence at its 3′-end. In some embodiments, the exemplaryoligonucleotides include one or more additional sequences. For example,a first oligonucleotide can include a functional domain (e.g., any ofthe variety of functional domains described herein). In someembodiments, two or more domains present in the exemplaryoligonucleotides each include a portion of a domain, such that when theexemplary oligonucleotides are joined, a full domain is generated. Forexample, a first oligonucleotide can include a first portion of a domain(e.g., a spatial barcode) and a second oligonucleotide can include asecond portion of a domain (e.g., a spatial barcode) such that thespatial barcode is generated upon joining the first and secondoligonucleotides.

FIGS. 31A-B depict at least one embodiment of a method for preparing anexemplary spatial array composition of the present disclosure. First, asshown in FIG. 31A, a substrate is provided that includes a firstoligonucleotide attached through its 5′-end to the substrate. In atleast one embodiment, the first oligonucleotide includes a sequence thatis identical across all of the plurality of the differentoligonucleotides attached to the substrate. In some embodiments, thefirst oligonucleotide can be understood as a common oligonucleotide“lawn” across the surface of the substrate that does not differ insequence in the various regions of the substrate. The free, unattachedend of the first oligonucleotide, thus, effectively acts as the startingpoint on the spatial array for the spatially-directed in situ synthesisof the second oligonucleotide that includes the spatial barcode.

In at least one embodiment, the length of the first oligonucleotide isthe same across the surface of the substrate. While typically notcritical to the function of the spatial array, in at least oneembodiment, the length of the first oligonucleotide sequence should beat least about 2 nucleotides in length. Furthermore, in some embodimentsthe length of the first oligonucleotide is at least about 5 nucleotidesin length, at least about 10 nucleotides in length, at least about 15nucleotides in length, at least about 20 nucleotides in length, at leastabout 25 nucleotides in length, at least about 27 nucleotides in length,at least about 30 nucleotides in length, at least about 33 nucleotidesin length, or at least about 35 nucleotides in length. Similarly, insome embodiments the first oligonucleotide has a length of about 2nucleotides in length to about 40 nucleotides in length, about 5nucleotides in length to about 40 nucleotides in length, about 10nucleotides in length to about 30 nucleotides in length, about 25nucleotides in length to about 35 nucleotides in length, about 20nucleotides in length to about 40 nucleotides in length, about 25nucleotides in length to about 40 nucleotides in length, about 25nucleotides in length to about 35 nucleotides in length, or about27nucleotides in length to about 33 nucleotides in length.

As further depicted in FIG. 31A, in at least one embodiment, the firstoligonucleotide can include phosphodiester and exocyclic amine moietiesthat are protected. Protection of the phosphodiester and exocyclic aminemoieties of the first oligonucleotide allows for optimal in situsynthesis of the second oligonucleotides that includes the spatialbarcode. Suitable protecting groups for phosphodiester and exocyclicamine moieties are described herein and are well-known in the art andinclude the protecting groups employed in standard oligonucleotidesynthesis using phosphoramidite chemistry.

The present disclosure describes a variety of embodiments for preparinga substrate having attached thereto a first oligonucleotide, wherein thefirst oligonucleotide includes protected phosphodiester and exocyclicamine moieties (see e.g., embodiments of FIGS. 32A-C to 36A-C).

In at least one exemplary embodiment, depicted in FIGS. 32A-C, themethod further includes preparing the first oligonucleotide ex situ(e.g., on a separate substrate or feature using solid-phase automatedsynthesis), cleaving it from the separate substrate or feature with theprotecting groups intact (see FIG. 32A), and subsequently attaching theex situ synthesized oligonucleotide with protecting groups it to thesubstrate (see FIG. 32B). A free hydroxyl at the unattached end isavailable for subsequent spatially-directed in situ synthesis of asecond oligonucleotide, e.g., a second oligonucleotide including aspatial barcode (see FIG. 32C).

In at least another exemplary embodiment, depicted in FIGS. 33A-C, themethod further includes preparing the first oligonucleotide in situdirectly on the substrate using base-by-base phosphoramidite synthesischemistry with phosphodiester and exocyclic amine protecting groups.Such a method can be use standard solid-phase oligonucleotide synthesischemistry to provide a substrate with first oligonucleotide attachedthereto by the 3′-end and having protecting groups. In at least oneembodiment, as depicted in FIG. 33A, oligonucleotide synthesis chemistryin a 5′-to-3′ direction can be carried out to provide the protectedfirst oligonucleotides attached to the substrate through the 5′-end. Inboth cases, the free hydroxyl at the unattached end is available forsubsequent spatially-directed in situ synthesis of a secondoligonucleotide, e.g., a second oligonucleotide including a spatialbarcode (see FIG. 32C).

In some embodiments, as depicted in FIG. 31B, a plurality of secondoligonucleotides of about 8-mer to about 30-mer are prepared byspatially-directed in situ synthesis extending from the free end of thefirst oligonucleotide. As shown in FIG. 31B, the spatially-directed insitu synthesis can be a light-directed spatial synthesis which iscarried out in the 5′-to-3′ direction. This in situ synthesis methodresults in a plurality of second oligonucleotide each attached throughits 5′-end to a 3′-end of a first oligonucleotide. FIG. 31B is aschematic representation of only two distinct regions each having fourindividual oligonucleotide molecules. Each region can have a differentsecond oligonucleotide sequence, and all of the second oligonucleotidemolecules in a region can have the same sequence, although as discussedfurther below, less than 100% yield in the in situ synthesis processresults in less than perfect purity (or homogeneity) of oligonucleotidesequence (e.g., a second oligonucleotide) in a region.

In at least one embodiment of the present disclosure, spatially-directedsynthesis of a plurality of oligonucleotides on a substrate in spatiallydistinct regions is carried out using light-directed synthesis or bymechanically-directed synthesis methods. A spatially-directed in situsynthesis method inherently correlates each sequence synthesized withthe location of synthesis. In the schematic illustration of FIG. 31B, alight-directed spatial synthesis is used to synthesize a secondoligonucleotide in a base-by-base step-wise fashion in each region. Theregion where each nucleotide base is added can be controlled by theprecise location (and duration) of spatially-directed illumination ofthe substrate. The direction and duration of illumination can beprecisely controlled, for example with maskless array synthesis (MAS)technology using computer-controlled digital micromirror devices (DMD),to provide distinct regions of 1 µm², or even smaller. It is only atthese precisely illuminated regions on the substrate where deprotectionof a photo-labile protecting group occurs, thereby allowing coupling ofa nucleoside phosphoramidite that provides the next base in theoligonucleotide sequence. In some embodiments, the synthesis of thefirst oligonucleotide is not light-directed synthesis.

In some embodiments, light-directed spatial in situ synthesis providesoptimal control in the synthesis of high-fidelity second oligonucleotidespatial barcode sequences at precise region locations on a substrate.Light-directed spatial synthesis of oligonucleotides refers to syntheticmethods using photolabile nucleoside protecting groups andspatially-directed illumination to effect selective deprotection andbase-by-base synthesis at specific locations on a substrate.Light-directed spatial synthesis of oligonucleotides uses an iterativecycle of phosphoramidite-based coupling chemistry with photo-labileprotecting groups rather than dimethoxytrityl (DMT) protecting groups.The synthesis cycle, thus, is very similar to that used in standardsolid-phase phosphoramidite synthesis of oligonucleotides with thefollowing general steps: (1) photo-deprotection; (2) activation andcoupling; (3) capping; and (4) drying. The drying step (4) can involveblowing helium to dry the substrate surface after coupling andacetonitrile wash, but before the next photodeprotection step.Techniques, protocols, reagents, materials, and other instrumentationuseful for light-directed spatial synthesis of high-densityoligonucleotide microarrays are described herein, are well-known in theart, and are commercially available. For example, US Pat. Nos.7,844,940, 8,030,477, PCT Publ. No. WO 99/63385A1, US Pat. Publ. No.20150246336A1, Agbavwe et al., Efficiency, error and yield inlight-directed maskless synthesis of DNA microarrays, Journal ofNanobiotechnology 9.1 (2011): 57; and Sack et al., Expressphotolithographic DNA microarray synthesis with optimized chemistry andhigh-efficiency photolabile groups, Journal of Nanobiotechnology 14.1(2016): 14, describe materials and methods that can be used in thelight-directed spatial synthesis methods of the present disclosure. Theaforementioned patents and publications are each incorporated herein byreference in their entireties.

In some embodiments, the photo-deprotection step involvesspatially-directed illumination of the substrate and is the step thatultimately defines the size and location of a region. Efficiency andaccuracy of the photo-deprotection step in defining the regions on thesubstrate where specific oligonucleotides are synthesized dependslargely on the illumination techniques and the photo-labile groups used.Photo-labile groups and illumination techniques useful forlight-directed spatial synthesis methods are described herein, arewell-known, and are commercially available (e.g., from Sigma-Aldrich,St. Louis, MO, USA). For example, photo-labile protecting groups thatcan be used to prepare spatial array compositions of the presentdisclosure are described in US Pat. Publ. No. 20150246336A1; Agbavwe etal., Efficiency, error and yield in light-directed maskless synthesis ofDNA microarrays Journal of Nanobiotechnology 9.1 (2011): 57; Kretschy etal., Next-Generation o-Nitrobenzyl Photolabile Groups for Light-DirectedChemistry and Microarray Synthesis, Angewandte Chemie InternationalEdition 54.29 (2015): 8555-8559; Sack et al., Express photolithographicDNA microarray synthesis with optimized chemistry and high-efficiencyphotolabile groups, Journal of Nanobiotechnology 14.1 (2016): 14;Lietard et al., High-Density RNA Microarrays Synthesized In Situ byPhotolithography, Angew. Chem. Int. Ed. 57 (2016): 15257-15261; and Hölzet al., High-Efficiency Reverse (5′→ 3′) Synthesis of Complex DNAMicroarrays, Scientific reports 8.1 (2018): 15099, each of which areincorporated herein by reference in their entireties.

In at least one embodiment, the photo-labile protecting groups useful inthe light-direct spatial synthesis methods of the present disclosureinclude NPPOC, Bz-NPPOC, and SPh-NPPOC which are shown in Table 2.

TABLE 2 Exemplary Photo-labile Protecting Groups Name Structure (e.g.,protecting 3′—OH of nucleoside) NPPOC

Bz-NPPOC

SPh-NPPOC

The schematic illustration of FIG. 31B depicts light-directed spatialsynthesis of second oligonucleotide in the 5′-to-3′ direction. Theresulting plurality of the second oligonucleotide are thus attachedthrough their 5′-ends to the free 3′-ends of the first oligonucleotideon the substrate. Accordingly, in at least one embodiment thelight-directed spatial synthesis of the second oligonucleotides iscarried out in the 5′-to-3′ direction. Techniques, reagents, andphoto-labile protecting groups useful for synthesis in the 5′-to-3′direction include those described in e.g., Hölz et al., High-EfficiencyReverse (5′→ 3′) Synthesis of Complex DNA Microarrays, Scientificreports 8.1 (2018): 15099, which is incorporated herein by reference. Inat least one embodiment, light-directed spatial synthesis of a pluralityof second oligonucleotides can be carried out using one or more of thephoto-labile protecting groups of Table 1 to protect the 3′—OH. In atleast one embodiment, the photo-labile protecting group used inlight-directed spatial synthesis of the second oligonucleotide isBz-NPPOC.

In at least one embodiment, the spatially-directed in situ synthesis canbe carried out using mechanically-directed spatial synthesis.Mechanically-directed spatial synthesis of oligonucleotides refers tomethods that use computer-controlled liquid dispensing and/or placementtechnology to mechanically direct nucleoside phosphoramidite reagents atdistinct locations on a substrate, and thereby synthesizing anoligonucleotide (e.g., a second oligonucleotide) using standardsolid-phase phosphoramidite chemistry at a distinct region on asubstrate. A range of technologies for mechanical spatially-directed insitu synthesis of oligonucleotides are described herein and arewell-known in the art.

The schematic illustration of FIG. 31B depicts only two regions, howeverit is contemplated that a spatial array composition made by the methodsof the present disclosure can include millions of different regions on asubstrate, each including a different second oligonucleotide spatialbarcode. In at least one embodiment, the number of secondoligonucleotides including different sequences is at least about100,000, at least about 250,000, at least about 500,000, at least about750,000, at least about 1,000,000, at least about 1,500,000, at leastabout 3,000,000, at least about 5,000,000, at least about 10,000,000, atleast about 100,000,000, at least about 500,000,000, or at least about1,000,000,000. In some embodiments, the number of second oligonucleotideincluding different sequences is about 100,000 to about 1,000,000,000,about 750,000 to about 1,500,000, about 1,000,000 to about1,000,000,000, about 1,000,000 to about 10,000,000, or about 1,500,000to about 5,000,000.

In order for a spatial array to accommodate these large numbers ofdifferent oligonucleotides on a substrate, it also is contemplated thatthe areas of the regions can be sufficiently small. Accordingly, in atleast one embodiment of the method of the present disclosure the regionsof the substrate have an area of less than about 2500 µm², less thanabout 1600 µm², less than about 400 µm², less than about 100 µm², lessthan about 50 µm², less than about 25 µm², less than about 15 µm², lessthan about 10 µm², less than about 5 µm², less than about 2 µm², lessthan about 1 µm², or smaller.

In at least one embodiment, methods of the present disclosurecontemplate the spatially-directed in situ synthesis of secondoligonucleotides including millions of different spatial barcodes eachin a different region. In order to accommodate millions of distinctregions each with a distinct spatial barcode, the present disclosurecontemplates that the second oligonucleotides have a length range ofabout 8 nucleotides to about 30 nucleotides. A length range of about 8nucleotides to about 30 nucleotides allows for the plurality of secondoligonucleotide to have extremely large number of distinctoligonucleotides that are indexed for their location on the substrate.For example, in the case of an 8 nucleotides sequence, there are 48 or65,536 different possible barcode sequences that can be used to identifydistinct locations on a substrate. In the case of a 12 nucleotidessequence, there are 412 or 16,777,216 different possible spatialbarcodes that can be used to identify distinct locations on a substrate.Accordingly, in at least one embodiment of the method of the presentdisclosure the second oligonucleotide have a length selected from 8nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28nucleotides, 29 nucleotides, or 30 nucleotides.

In order to provide for optimal functionality of a spatial array, thecorrelation of the spatial barcodes with the locations should be high.Accordingly, the spatially-directed in situ synthesis of the spatialbarcode should result in high fidelity or purity of spatial barcode ineach of the regions. It is an advantage of the spatially-directedsynthesis methods of the present disclosure that the secondoligonucleotide are prepared on a substrate with high fidelity.Generally, the fidelity (or sequence purity) of oligonucleotidesequences prepared by step-wise solid-phase synthesis decreases as thelength of the oligonucleotide being synthesized increases. For example,assuming a spatially-directed in situ synthesis having a couplingefficiency of 99.5%, the fidelity of 12 nucleotide barcode sequences ina region is ~95%. In some embodiments (e.g., as in FIGS. 32A-C), the useof spatially-directed in situ synthesis to prepare only the 8 to 30nucleotide second oligonucleotides results in spatial barcodes ofhigh-fidelity. Accordingly, in at least one embodiment of the methods ofthe present disclosure the resulting second oligonucleotide synthesizedin each of the different regions of the substrate has a purity of atleast 75%, at least 80%, at least 85%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or more.

In at least one embodiment, the second oligonucleotide is unique foreach region of the substrate. Additionally or alternatively, in someembodiments, the second oligonucleotide spatial barcode includes asequence that uniquely identifies it from the plurality of other spatialbarcode sequences on the spatial array but does not interfere with thecapture sequences (e.g., capture domains) of the oligonucleotides (e.g.,capture probes) that target binding to nucleic acids in a sample. Uniquespatial barcodes can be generated using random sequence generation,along with stringent filtering of sequences by mapping to the genomes ofall common reference species using desired Tm intervals, and GC content,to minimize interference with capture sequence (e.g., capture domain)binding to analytes, and to optimize the difference from the otherspatial barcodes. Thus, in at least one embodiment, the spatial barcodesof the spatial array are selected such that desired analytes of thebiological sample applied to the array do not hybridize specifically tothe spatial barcode of the second oligonucleotide. For example, in someembodiments, the spatial barcode of the spatial array have less than 80%sequence complementarity to the targeted nucleic acid sequences (e.g.,analytes) in the sample applied to the spatial array. In someembodiments, spatial barcode has less than 70% sequence idcomplementarity entity, less than 60% sequence complementarity, lessthan 50% sequence complementarity, or less than 40% sequencecomplementarity to the nucleic acids in the biological sample targetedfor capture by the plurality of oligonucleotide sequences.

In some embodiments, as depicted in FIG. 31C, a plurality of thirdoligonucleotides are attached to the unattached end of the secondoligonucleotide. In at least one embodiment, the third oligonucleotideis in the same orientation as the second oligonucleotide. Thus, if thesecond oligonucleotide is attached to the first oligonucleotide throughits 5′-end (in a 5′-to-3′ orientation relative to the substrate) thenthe third oligonucleotide is attached through its 5′-end to the 3′-endof the second oligonucleotide. As exemplified in FIG. 31C, in at leastone embodiment, the third oligonucleotides are attached through their5′-ends to the 3′-ends of the plurality of second oligonucleotides. Insome embodiments, the third oligonucleotide has a length of about 5 toabout 40 nucleotides and includes a capture sequence (e.g., capturedomain) capable of hybridizing to an analyte (e.g., nucleic acid) in abiological sample. As described further herein, in some embodiments, thethird oligonucleotide includes a free 3′—OH and is capable ofhybridizing to a complementary nucleic acid (e.g., an analyte) in abiological sample and then undergoing biosynthetic (e.g., polymerasecatalyzed) extension from it 3′—OH.

In at least one embodiment, the plurality of third oligonucleotides havea length of at least about 5 nucleotides, at least about 10 nucleotides,at least about 15 nucleotides, at least about 20 nucleotides, at leastabout 27 nucleotides, at least about 30 nucleotides, at least about 33nucleotides, or at least about 35 nucleotides. In some embodiments, theplurality of third oligonucleotide have a length of about 5 nucleotidesto about 40 nucleotides, about 10 nucleotides to about 40 nucleotides,about 20 nucleotides to about 40 nucleotides, about 25 nucleotides toabout 35 nucleotides, or about 27 nucleotides to about 33 nucleotides.

In at least one embodiment of the methods of the present disclosure, thethird oligonucleotides are prepared off the substrate and attached as awhole segment by chemical coupling or ligation to the plurality ofsecond oligonucleotides. In at least one embodiment, the plurality ofthird oligonucleotides are attached by ligation. In some embodiments,protected moieties of the first and second oligonucleotides aredeprotected prior to ligating the plurality of third oligonucleotides.

In at least one embodiment where the method of the present disclosurefurther includes attaching a plurality of third oligonucleotides, thefirst oligonucleotide is attached to the substrate through its 5′-end,the plurality of second oligonucleotides are prepared byspatially-directed in situ synthesis on the 3′-end of the firstoligonucleotide, and the plurality of third oligonucleotides areattached through their 5′-end to the 3′-end of the plurality of secondoligonucleotides.

Alternatively, in at least one embodiment where the method of thepresent disclosure further includes attaching a plurality of thirdoligonucleotides, the first oligonucleotide can be attached to thesubstrate through its 3′-end, the plurality of second oligonucleotidesare prepared by spatially-directed in situ synthesis on the 5′-end ofthe first oligonucleotide, and the plurality of third oligonucleotidesare attached through their 3′-end to the 5′-end of the plurality ofsecond oligonucleotides.

In at least one embodiment, the third oligonucleotide includes a capturesequence (e.g., a capture domain) capable of hybridizing to a nucleicacid sequence (e.g., an analyte) in a biological sample. In at least oneembodiment, the capture sequence is located at the free 3′-end of thethird oligonucleotide which is oriented with its 5′-end attached to thesecond oligonucleotide. In this orientation the free 3′-end can beextended (e.g., by template dependent polymerization). It iscontemplated that in at least one embodiment, the capture sequence(e.g., capture domain) of the third oligonucleotide includes a sequencecapable of hybridizing to a specific type of target nucleic acid that ispresent in a biological sample. For example, the capture sequence of thethird oligonucleotide can be designed to selectively capture mRNA byhybridization to the poly-A tail. Accordingly, in at least oneembodiment, the capture sequence of the third oligonucleotide includes apoly-T oligonucleotide. In some embodiments, the capture sequence caninclude nucleotides which are functionally analogous to poly-T andcapable of binding (e.g., hybridizing) selectively to poly-A, such as apoly-U oligonucleotide. In at least one embodiment, the capture sequenceof the third oligonucleotide includes a poly-T or poly-U sequence of atleast about 5 nucleotides to about 40 nucleotides in length. In someembodiments, the capture sequence (e.g., capture domain) includes apoly-T or poly-U sequence of at least 5 nucleotides, 10 nucleotides, atleast 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides,at least 30 nucleotides, at least 35 nucleotides, or at least 40nucleotides in length.

In some embodiments, it is contemplated that the capture sequence can bedesigned to selectively bind to a desired sub-type or subset of nucleicacid targets suspected of being present in a biological sample. Forexample, a particular type of RNA, such as mRNA, microRNA or rRNA, or aparticular mRNA species that corresponds to a particular group of genes(e.g., gene specific capture domain). Thus, the capture sequence of thethird oligonucleotide may include a sequence that specifically binds toa particular target gene or set of genes, and can be based on aparticular sequence motif sequence or conserved sequence according tomethods well known in the art.

In some embodiments, the third oligonucleotides of the plurality of thethird oligonucleotides can include a random sequence. As is well-knownin the art, such random sequences (e.g. random hexamers or nonamers) areeffective for the capture nucleic acid targets (e.g., analytes) byselective binding. Accordingly, it is contemplated that the capturesequence (e.g., capture domain) of the third oligonucleotides of theplurality of third oligonucleotides can include a random sequence, wherethe random sequences are at least 6-mers, at least 8-mers, at least10-mers, at least 12-mers, at least 14-mers, at least 16-mers, orlonger. In some embodiments, the plurality of third oligonucleotides caninclude random sequences together with a poly-T or poly-U capturesequence. Accordingly, where a third oligonucleotide includes a poly-Tor poly-U capture sequence, it can also include a random sequence. Insome embodiments, where the third oligonucleotide is in a 5′-to-3′orientation, the random sequences are located at the 5′-end of the thirdoligonucleotide adjacent to the 3-end of the second oligonucleotideincluding the spatial barcode.

In at least one embodiment, the first, second, and thirdoligonucleotides are linked via phosphodiester bonds. Additionally, insome embodiments, it is contemplated that the oligonucleotides can beattached via non-natural oligonucleotide linkages, such asmethylphosphonate or phosphorothioate bonds, or via other non-naturalbiocompatible linkages such as click-chemistry linkages. The bondslinking the oligonucleotides can be formed by synthetic phosphoramiditecoupling chemistry, by a synthetic click-chemistry described herein, bya ligation reaction, by enzymatic biosynthesis of nucleic acid polymers(e.g., using a polymerase or transcriptase), or a combination of bondforming reactions. For example, in at least one embodiment, the secondoligonucleotide is attached to the first oligonucleotide by standardphosphoramidite chemistry forming a phosphodiester linkage, and thethird oligonucleotide is attached to the unattached end of the secondoligonucleotide via a ligation reaction.

The plurality of first oligonucleotides on a spatial array can beattached (e.g., covalently bound or immobilized) to the substrate viaeither the 3′-end or 5′-end of the oligonucleotide. The attachment of anoligonucleotide to a substrate of a spatial array through a covalentbond can be either direct (e.g., substrate to 3′-end of oligonucleotide)or indirect (e.g., substrate to linker molecule to 3′-end ofoligonucleotide). Typically, for commercially available DNA microarrays,the oligonucleotides are attached via a 3′ linkage, thereby leaving afree 5′-end. The free 5′-end can be used for further synthesis on theoligonucleotide strand. However, oligonucleotides attached to thesubstrate via the 5′-end, provide an advantage in having their 3′-endsavailable for further biosynthetic extension (e.g., using polymerase ortranscriptase) as described elsewhere herein.

In at least one embodiment, the first oligonucleotide is attached to thesubstrate through its 5′-end, the second oligonucleotide is attached tothe 3′-end of the first oligonucleotide through its 5′-end, and thethird oligonucleotide is attached to the 3′-end of the secondoligonucleotide through its 5′-end.

In at least one embodiment, the first oligonucleotide is attached to thesubstrate through its 3′-end; the second oligonucleotide that isattached to the 5′-end of the first oligonucleotide through its 3′-end,and the third oligonucleotide is attached to the 5′-end of the secondoligonucleotide through its 3′-end.

In at least one embodiment of the composition of the present disclosurethe length of the oligonucleotide (e.g., a first oligonucleotide, asecond oligonucleotide, and/or a third oligonucleotide) in each regionis at least 10 nucleotides, at least 20 nucleotides, at least 30nucleotides, at least 40 nucleotides, at least 50 nucleotides, or atleast 60 nucleotides. In some embodiments the length of theoligonucleotide in each region is from 5 nucleotides to 100 nucleotides,from 10 nucleotides to 100 nucleotides, from 20 nucleotides to 100nucleotides, from 40 nucleotides to 100 nucleotides, from 50 nucleotidesto 90 nucleotides, or from 60 nucleotides to 80 nucleotides.

As described elsewhere herein, in some embodiments of the spatial arraysof the present disclosure, the first oligonucleotide of the spatialarray includes a 2-mer to 40-mer having the common sequence attachedacross all, or a substantial contiguous portion, of the surface of thesubstrate. Thus, the step of attaching the common first oligonucleotideto the substrate does not need to be spatially directed. Accordingly, avariety of different embodiments can be used to attach prepare asubstrate having a common first oligonucleotide of 2 to 40 nucleotidesin length attached thereto. In some embodiments, the firstoligonucleotide of the spatial array can include various domainsdescribed herein. For example, the first oligonucleotide can include acleavage domain, a functional domain (e.g., sequencing handle), a uniquemolecular identifier, or combinations thereof.

As illustrated by FIGS. 32A-C, in at least one embodiment, a firstoligonucleotide can be prepared ex situ by standard automatedsolid-phase oligonucleotide synthesis in a 3′-to-5′ direction usingphosphoramidite chemistry. However, as described elsewhere herein,protection of the phosphodiester and exocyclic amine moieties of thefirst oligonucleotide facilitates the spatially-directed in situsynthesis of the second oligonucleotide on the unattached end of thefirst oligonucleotide. Thus, as shown in FIG. 32A, in at least oneembodiment, the first oligonucleotide is synthesized ex situ usingstandard techniques, however, the synthesized oligonucleotide is cleavedfrom the substrate without deprotecting the phosphodiester and exocyclicamine moieties that are protected during synthesis. The protected commonoligonucleotide used for the first oligonucleotide is attached to thesubstrate with the protecting groups intact as shown in FIG. 32B.

The standard oligonucleotide synthesis method using phosphoramiditechemistry was developed in the 1980s and later enhanced with solid-phasesupports and automation. It the method used by commercial synthesis ofDNA and RNA oligonucleotides. Generally, solid-phase oligonucleotidesynthesis proceeds in the 3′ → 5′ direction rather than 5′ → 3′direction of natural biosynthesis. The solid-phase phosphoramiditesynthesis cycle generally includes four steps: (1) detritylation; (2)coupling; (3) oxidation; and (4) capping. Briefly, the four steps in thesynthesis cycle are as follows. In step (1), “detritylation,” a 5′-DMTprotecting group is removed from the first, solid-support-linkednucleoside. In step (2), “coupling,” the de-protected 5′—OH of thefirst, solid-support-linked nucleoside attacks the phosphorus of theincoming second nucleoside to form a phosphite triester. In step (3),“oxidation,” the relatively unstable phosphite triester is converted toa stable phosphate triester. In step (4), prior to moving to step (1) ofthe next cycle, “capping,” is carried out wherein unreacted 5′—OH groupsare acetylated, thereby preventing elongation of sequences with deletionmutations.

In at least one embodiment of the ex situ synthesis method, the finalmonomer unit added to the 5′-end during ex situ synthesis of the firstoligonucleotide can include a non-natural nucleoside that facilitatesattachment or immobilization of the first oligonucleotide to thesubstrate or linkers attached to the substrate via its 5′-end.Alternatively, in some embodiments of the ex situ synthesis method, thefirst monomer unit added to the 3′-end during ex situ synthesis of thefirst oligonucleotide can include a non-natural nucleoside that uponcleavage from the synthesis substrate or feature can facilitateattachment or immobilization of the first oligonucleotide to thesubstrate or linkers attached to the substrate via the 3′-end.

In at least one embodiment, the first oligonucleotide attached to thesubstrate is prepared by in situ synthesis on the substrate. Generally,because the sequence of the first oligonucleotide is the same across allregions of the substrate, it is not necessary to use spatially-directedin situ synthesis in such an embodiment. For example, in situ synthesisof the first oligonucleotide can be carried out on the substrate usingstandard solid-phase phosphoramidite chemistry and reagents. As notedabove, maintaining protection of the phosphodiester and exocyclic aminemoieties of the first oligonucleotide facilitates the spatially-directedin situ synthesis of the second oligonucleotide on the unattached end ofthe first oligonucleotide. Accordingly, in at least one embodiment, itis contemplated that the in situ synthesis first oligonucleotide on thesubstrate is carried out without the final step of deprotecting thephosphodiester and exocyclic amine moieties before the secondoligonucleotide is synthesized.

FIGS. 33A-B illustrate an exemplary embodiment of a method for in situsynthesis of a common first oligonucleotide directly on the substrate.The synthesis can be carried out using step-wise solid-phasephosphoramidite chemistry but without final cleavage or deprotectionsteps. As shown in FIG. 33A, in this embodiment, the synthesis wouldresult in a first oligonucleotide attached across the surface of thesubstrate with protected phosphodiester and exocyclic amine moieties.Following this in situ synthesis of the first oligonucleotide withprotective groups intact, light-directed spatial synthesis of the secondoligonucleotide to the 3′-ends of the first oligonucleotides can becarried out as shown in FIG. 33B.

In at least one embodiment, the first oligonucleotide attached to thesubstrate is prepared by spatially-directed in situ synthesis on thesubstrate. The spatially-directed oligonucleotide synthesis method usedcan include light-directed spatial synthesis and/ormechanically-directed spatial synthesis described elsewhere herein. Inthis embodiment, the spatially-directed synthesis can be used inessentially the same way as the in situ standard oligonucleotidesynthesis for the preparation of the first oligonucleotide having asingle sequence across all regions of the substrate. It is contemplated,however, that in some embodiments spatially-directed synthesis can beused to prepare a substrate having the first oligonucleotide attached inselected regions of the substrate. For example, in some embodiments, aspatial array could be prepared having more than one firstoligonucleotide sequence in different locations of the substrate.

Accordingly, as shown in FIGS. 34A-B that in at least one embodiment thecommon first oligonucleotide can be in situ synthesized directly on thesubstrate however using light-directed synthesis with photolabilegroups, rather than standard protective groups, such as dimethoxytritylgroups. In some embodiments, the synthesized first oligonucleotideswould be prepared using light-directed deprotection but would not becleaved from the substrate or deprotected as shown in FIG. 34A.Following this light-directed in situ synthesis of the firstoligonucleotide with protective groups intact, light-directed spatialsynthesis of the second oligonucleotide to the 3′-ends of the firstoligonucleotides can be carried out as shown in FIG. 34B.

Generally, during standard automated oligonucleotide synthesis,following cleavage of the oligonucleotide from the synthesis substrateor feature, the protecting groups on the phosphates and bases areremoved (or deprotected) by treatment in concentrated aqueous ammoniawith heat. Accordingly, it is also contemplated in at least oneembodiment that a first oligonucleotide is synthesized ex situ usingstandard automated synthesis, including cleavage and deprotection of thephosphodiesters and/or exocyclic amine moieties. As illustrated by theschematic of FIG. 35A, the cleaved and deprotected firstoligonucleotides synthesized ex situ can be attached to the substratesurface through a linkage to the 5′-end as described elsewhere herein.Then, as depicted by FIG. 35B, the phosphodiester and/or exocyclic aminemoieties can be protected in situ on the substrate. Following this insitu protection step, light-directed spatial synthesis of the secondoligonucleotide to the 3′-ends of the first oligonucleotides can becarried out as shown in FIG. 35C.

In an embodiment illustrated by the schematic of FIG. 36A, ex situsynthesized first oligonucleotides can be cleaved from their synthesissubstrate or feature, deprotected, and then attached to a substratesurface through a linkage to the 5′-end as described elsewhere herein.Then, as depicted by FIG. 36B, a complementary oligonucleotide can behybridized to the common first oligonucleotides located across thesubstrate. The formation of a doublestranded structure by hybridizationcan effectively protect the phosphodiester and/or exocyclic aminemoieties of the first oligonucleotide on the substrate. Following thisin situ protection-by-hybridization step, light-directed spatialsynthesis of the second oligonucleotide to the 3′-ends of the firstoligonucleotides can be carried out as shown in FIG. 36C.

In at least one embodiment, the spatial array compositions preparedusing the methods of the present disclosure can be useful for spatialtranscriptomics applications as described in e.g., U.S. Patent App. No.US2014/0066318A1, incorporated by reference herein in its entirety.Briefly, in spatial transcriptomics a complex multi-cellular sample,such as a biological sample (e.g., tissue section), is immobilized on aspatial array of oligonucleotides. Typically, the oligonucleotidesequences are attached to the array substrate through their 5′-end andinclude an mRNA capture sequence at their 3′-end. Each of theoligonucleotides also includes 5′-adjacent to the capture sequence, arandom primer sequence of at least 16-mer that allows for amplificationof any captured sequences. The detection process results inamplification of the captured mRNA sequence as well as the spatialbarcode. Sequencing of the amplicons thereby indicates not only theidentity of the transcribed gene but also the location in themulti-cellular sample where the gene was transcribed.

Furthermore, in order to survey the genes undergoing transcription in acomplex multi-cellular biological sample, the spatial array can includean extremely high density of different oligonucleotides specificallylocated on the substrate. For example, it is contemplated that spatialarrays useful in various applications, such as spatial transcriptomics,can include 1.5 million, 10 million, 100 million, and even up to 1.0billion different oligonucleotides, each of which has a differentspatial barcode sequence and is located in a different region on asubstrate, wherein the regions are less than 5 µm², even as small as 1.0µm².

As noted elsewhere herein, oligonucleotides of a spatial array caninclude a different spatial barcode that correlates with its location.In some embodiments, it is contemplated that additional oligonucleotidescan be present on the same substrate as the spatial array that include asequence identical to a barcode sequence of the spatial array. Suchadditional oligonucleotides can arise due to errors in synthesis or canbe prepared intentionally on the substrate (e.g., for calibration orquality control purposes). Generally, these sequences located in adifferent location on the same substrate are not considered part of thespatial array and are kept to a level and/or location that avoidsinterference with the intended function of the spatial array.

In some embodiments, methods provided herein can be used to generate anarray that allows resolution equal to or less than 10 µm, allowingsingle-cell resolution. In some embodiments, a spatial array isgenerated by providing a plurality of sets of primers on a substrate. Insome embodiments, a set of the sets of primers includes a first primerand a second primer present at a 1:1 ratio. In some embodiments, thesecond primer has a sequence that is complementary to the first primeror to a template oligonucleotide.

In some embodiments, methods of preparing a spatial array providedherein include providing a substrate, immobilizing a plurality of setsof primers on the substrate, providing a template oligonucleotide havinga reverse complement of a spatial barcode domain, a reverse complementof a capture domain, a first hybridization domain capable of hybridizingto the first primer, and a second hybridization domain, wherein areverse complement of the second hybridization domain is capable ofhybridizing to the second primer; hybridizing the templateoligonucleotide to the first primer; extending the first primer usingthe template oligonucleotide as a template to generate a capture probecomprising the first primer, the spatial barcode, and the capturedomain, which capture probe is immobilized on the substrate; andreleasing the template oligonucleotide from the spatial array.

In some embodiments, the spatial array comprises a plurality of sets ofprimers affixed to the substrate. In some embodiments, a first primerand a second primer that are at a 1:1 ratio or about at a 1:1 ratio(e.g., at or about a 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1,1.3:1, 1.2:1, 1.1.:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6,1:1.7, 1:1.8, 1:1.9, or 1:2 ratio) and that are less than 10 µm apart onthe substrate. In some embodiments, the first primer and the secondprimer are about 0.1 µm, 0.2 µm, 0.3 µm, 0.4 µm, 0.5 µm, 0.6 µm, 0.7 µm,0.8 µm, 0.9 µm, 1.0 µm, 2.0 µm, 3.0 µm, 4.0 µm, 5.0 µm, 6.0 µm, 7.0 µm,8.0 µm, 9.0 µm, 10.0 µm, 15 µm, 20 µm, 25 µm, 30 µm, 35 µm, 40 µm, 45µm, 50 µm, 55 µm, 60 µm, 65 µm, 70 µm, 75 µm, 80 µm, 85 µm, 90 µm, 95µm, or 100 µm apart on the substrate.

In some embodiments, the first primer and the second primer are about0.1 µm to about 100 µm apart on the substrate. In some embodiments, thefirst primer and the second primer are about 0.5 µm to about 50 µm aparton the substrate. In some embodiments, the first primer and the secondprimer are about 0.7 µm to about 25 µm apart on the substrate. In someembodiments, the first primer and the second primer are about 1.0 µm toabout 20 µm apart on the substrate. In some embodiments, the firstprimer and the second primer are about 1.0 µm to about 15 µm apart onthe substrate. In some embodiments, the first primer and the secondprimer are about 1.0 µm to about 10 µm apart on the substrate. In someembodiments, the first primer and the second primer are about 1.0 µm toabout 5 µm apart on the substrate. In some embodiments, the first primerand the second primer are about 1.0 µm to about 2 µm apart on thesubstrate.

In some embodiments, the first primer is affixed to the substrate at the5′ end of the first primer. In some embodiments, the second primer isaffixed to the substrate at the 5′ end of the second primer.

In some aspects, the first primer is single stranded DNA. In someaspects, the first primer is RNA. In some aspects, the second primer issingle stranded DNA. In some aspects, the second primer is RNA.

In some embodiments, the first primer on the substrate comprises asequence that is complementary to the template oligonucleotide andallows the template oligonucleotide to bind to the first primer. In someembodiments, the first primer can also comprise a poly (A) or poly (T)domain that is complementary to a poly(T) or poly(A) domain of thesecond primer. In some embodiments, the first primer can also comprise acleavage domain. In some embodiments, the second primer can alsocomprise a cleavage domain.

In some embodiments, the first primer and the second primer are used toamplify a template oligonucleotide to create a cluster of one or morecapture probes. In some aspects, the template oligonucleotide includes areverse complement of a unique molecular identifier (UMI).

In some embodiments, the template oligonucleotide is attached to a bead(e.g., any of the variety of beads described herein, e.g., a gel bead).In some embodiments, the template oligonucleotide is attached to thebead at the 3′ end. In some embodiments, the template oligonucleotide isattached to the bead at the 5′ end. In some embodiments, templateoligonucleotides that have a bead at the 3′ end also have 5′modification, which allows the template oligonucleotide to attach to asupport (e.g., any of the variety of functionalized groups for attachingnucleic acids to substrates, e.g., slides, described herein or otherwiseknown in the art). In some embodiments, template oligonucleotides thathave a bead at the 5′ end also have 3′ modification, which allows thetemplate oligonucleotide to attach to a support (e.g., any of thevariety of functionalized groups for attaching nucleic acids tosubstrates, e.g., slides, described herein or otherwise known in theart). In some aspects, the template oligonucleotide comprises a cleavagesequence that allows the template oligonucleotide to detach from thebead (e.g., after the oligonucleotide has been attached to the support).

In another embodiment, the primer on the support has a non-barcodeduniversal primer that hybridizes to the template oligonucleotide onbeads. In some embodiments, after one round of synthesis, the templateoligonucleotide (e.g., a barcode on the template oligonucleotide) isincorporated.

In some aspects, the bead is a gel bead. In some aspects, the bead isless than 10 µm in diameter. In some aspects, the bead is less than 5 µmin diameter. In some aspects, the bead is 2 µm in diameter. In someaspects, the bead is less than 2 µm in diameter. In some aspects, thebead is 1 µm in diameter. In some aspects, the bead is less than 1 µm indiameter.

In some aspects, after the template oligonucleotide binds to a primer,the bead is removed. In some aspects, the bead is removed prior toamplification of the template oligo. In some aspects, the bead isremoved after amplification of the template oligo. In some embodiments,the bead is removed by a washing step. Removal of beads can be performedusing techniques known in the art.

In some aspects, after binding of the template oligonucleotide to thefirst primer, the sequence of the first primer is extended. In someaspects, one or more beads (e.g., all of the beads or substantially allof the beads) that are attached to the template oligonucleotide(s) isremoved after extension of the first primer. In some aspects, the one ormore beads (e.g., all of the beads or substantially all of the beads)that are attached to the oligonucleotide(s) is removed before extensionof the primer.

In some aspects, the one or more beads is non-spherical. In someaspects, the non-spherical bead or one or more non-spherical beads isprimer loaded. In some aspects, the non-spherical bead or one or morenon-spherical beads is hexagonal. In some aspects, hexagonal beads canform in a honeycomb pattern when placed on a support. In some aspects,the non-spherical bead or one or more non-spherical beads is cuboidal.In some aspects, one or more hexagonal or cuboidal beads allows for morecompact packing of beads on a substrate than would occur with sphericalbeads. In some embodiments, after initial binding of the templateoligonucleotide and a first primer, a capture probe is generated. Insome aspects, the capture probe is generated using cluster expansion.

In some aspects, the capture probe is generated using bridgeamplification. The term “bridge amplification” refers to a method ofamplifying nucleic acid in which primers (e.g., a first primer and asecond primer) that are extended in the reaction are tethered to asubstrate by their 5′ ends. In some embodiments, the process is repeatedto provide amplified, immobilized nucleic acid molecules.

In some embodiments, bridge amplification comprises a first stepcomprising hybridization of the target nucleic acid to the first primerattached to the support. A first amplification product, which iscomplementary to the target nucleic acid, is then formed by extension ofthe first primer sequence. On subjecting the support to denaturationconditions the target nucleic acid is released and can then participatein further hybridization reactions with other first primer sequenceswhich may be attached to the support. The first amplification productwhich is attached to the support, may then hybridize with the secondprimer attached to the support, forming a “bridge.” In some embodiments,a second amplification product comprising a nucleic acid sequencecomplementary to the first amplification product can be formed byextension of the second primer sequence and is also attached to thesupport. Thus, the target nucleic acid and the first and secondamplification products are capable of participating in a plurality ofhybridization and extension processes, limited only by the initialpresence of the target nucleic acid and the number of first primer andsecond primer sequences initially present and the result is a number ofcopies of the target sequence attached to the surface. Bridgeamplification is further disclosed U.S. Appls. Nos. US-2008-0160580 andUS-2008-0286795, each of which is incorporated by reference in itsentirety. In some aspects, the capture probe includes a UMI (e.g., anyof the variety of UMIs described herein). In some aspects, the captureprobe includes the sequence of the first primer, a spatial barcode(e.g., any of the variety of spatial barcodes described herein), and/ora capture domain (e.g., any of the variety of capture domains describedherein). In some aspects, the capture probe is immobilized on thesubstrate. In some aspects, the capture domain comprises a sequencecomplementary to an analyte of interest. In some aspects, the capturedomain comprises a sequence designed to capture messenger RNA. In someaspects, the capture domain comprises a poly(T) domain. In some aspects,the capture domain comprises a target-specific sequence (e.g., asequence complementary to the sequence of a target of interest, e.g., atarget nucleic acid molecule, e.g., a specific messenger RNA or DNA). Insome aspects, the capture domain comprises a random sequence (e.g. ahexamer).

In some embodiments, after a copy of the capture probe is generated, thecopy of the template oligonucleotide is released by cleaving thecleavage domain after the copy of the capture probe is generated.

In some embodiments, after cluster amplification, all or a portion ofthe sequence (e.g., identity) of the generated capture probe isdetermined. In some aspects, the all or a portion of the sequence (e.g.,identity) of the generated capture probe is determined after thetemplate oligonucleotide is released. In some aspects, the all or aportion of the sequence (e.g., identity) of the generated capture probeis determined in situ. In some aspects, the all or a portion of thesequence (e.g., identity) of the generated capture probe is determinedby in situ sequencing. As non-limiting examples, in situ sequencing canbe performed via sequencing-by-synthesis (SBS), sequential fluorescencehybridization, sequencing by ligation, nucleic acid hybridization, orhigh-throughput digital sequencing techniques.

In some embodiments, after the probe is clonally expanded, spatialanalysis of a biological analyte in a biological sample can beperformed. For example, spatial analysis can include determining alocation of an analyte in a biological sample. In some aspects, methodsof performing spatial analysis of a biological analyte in a biologicalsample include preparing the spatial array (e.g., via any methoddiscussed herein). In some aspects, the methods further include decodingthe one or more capture probes by determining the identity of thespatial barcode of the one or more capture probes, thereby associatingthe spatial barcode of the one or more capture probe with a location onthe spatial array; contacting the biological sample to the spatial arrayunder conditions wherein the biological analyte binds a capture probepresent in a cluster; extending the capture probe via a polymerizationreaction using the biological analyte as a template to generate anextended capture probe that include the sequence of the capture probeand the reverse complement of the sequence of the biological analyte;determining the sequence of the extended capture probe; and associatingthe spatial barcode of the capture probe with the captured biologicalanalyte, thereby determining spatial location of the analyte in thebiological sample.

Generating Capture Probes

Capture probes described herein can contain various functional sequencesand a capture domain. In some embodiments, the capture domain capturesanalytes in an unbiased manner. For example, if the capture domain is apoly(T) sequence, the capture domain can interact (e.g., hybridize) tothe poly(A) tail of any given mRNA present in the biological sample,regardless of the identity of that mRNA. In some embodiments, thecapture domain can capture analytes in an unbiased manner with via acapture domain having a random or degenerate sequence. As non-limitingexamples, a capture domain can include a random hexamer or octamersequence for unbiased capture. In some embodiments, the capture domaincaptures analytes in a biased manner. For example, the capture domaincan be gene specific, such that a specific analyte (e.g., a specificnucleic acid, e.g., a specific mRNA or DNA sequence) or set of analytes(e.g., set of nucleic acids, e.g., a set of mRNAs or a set of DNAs,e.g., a set of mRNAs or a set of DNAs sharing a common or similarsequence) can be captured for spatial analysis in the biological sample.In some embodiments, the capture domain can capture rRNA analytes. Forexample, the capture domain can contain a degenerate sequence capable ofinteracting (e.g., hybridizing) with 16 s rRNA. In some embodiments, thecapture domain can interact with analytes containing V(D)J sequences. Insome embodiments, the capture domain can interact with a capture agentbarcode domain associated with an analyte capture agent (e.g., ananalyte capture sequence). Capture domains for both unbiased analytecapture and biased capture can be generated from an existing array witha ligation strategy.

Capture probes can be generated from existing arrays with a ligationstrategy. In some embodiments, an array containing a plurality ofoligonucleotides (e.g., in situ synthesized oligonucleotides) can bemodified to generate a variety of capture probes. The oligonucleotidescan include various domains such as, spatial barcodes, UMIs, functionaldomains (e.g., sequencing handle), cleavage domains, and/or ligationhandles. As used herein, the term “ligation handle” refers to a portionof an oligonucleotide that is complementary to a portion of a differentoligonucleotide. In some embodiments, the different oligonucleotide is asplint oligonucleotide having a sequence complementary to a donoroligonucleotide, and the donor oligonucleotide is ligated to theligation handle. In some embodiments, the donor oligonucleotide includesa capture domain, such that upon ligation to oligonucleotide having aligation handle, a capture probe is generated. For example, FIG. 41shows an acceptor oligonucleotide on a immobilized substrate (e.g., anarray), that contains from 5′ to 3′ an amino modifier C6, followed by 12uracils that can function as a cleavage domain, a sequencing handle(e.g., R1), a 16 base pair spatial barcode, a 12 base pair UMI, and aligation handle. In some embodiments, a splint oligonucleotide with asequence complementary to the ligation handle of the acceptoroligonucleotide and an additional sequence complementary to anoligonucleotide (e.g., a donor oligonucleotide) containing a capturedomain (e.g., a capture domain of interest) facilitates the ligation ofthe oligonucleotide and the oligonucleotide containing the capturedomain. In some embodiments, the additional sequence complementary tothe capture domain is a poly(A) sequence. In some embodiments, theadditional sequence complementary to the capture domain is a sequencespecific to a nucleic acid (e.g., an mRNA or DNA sequence). In someembodiments, the additional sequence complementary to the capture domainis a sequence specific to a subset of nucleic acids (e.g., a subset ofmRNA or DNA sequences sharing a common or similar sequence). Inembodiments where the splint oligonucleotide sequence complementary tothe capture domain is a poly(A) sequence, the splint oligonucleotide canfacilitate the ligation of a poly(T) capture domain to theoligonucleotide. In embodiments where the splint oligonucleotidesequence complementary to the capture domain is a sequence specific to anucleic acid, the splint oligonucleotide can facilitate the ligation ofthat capture domain to the oligonucleotide. In some embodiments, thegene specific capture domain is for Penk mRNA. In embodiments where thesplint oligonucleotide sequence complementary to the capture domain is asequence specific to a subset of nucleic acids, the splintoligonucleotide can facilitate the ligation of that capture domain tothe oligonucleotide.

Capture probes can be generated from existing arrays with a ligationstrategy. In some embodiments, an array containing a plurality ofoligonucleotides (e.g., in situ synthesized oligonucleotides) can bemodified to generate a variety of capture probes. The oligonucleotidescan include various domains such as, spatial barcodes, UMIs, functionaldomains (e.g., sequencing handle), cleavage domains, and/or ligationhandles. For example, FIG. 44 shows an exemplary strategy where anacceptor oligonucleotide having a ligation handle is immobilized on asubstrate (e.g., an array). In some embodiments, the acceptoroligonucleotide shown in FIG. 44 contains from 5′ to 3′ one or more of(e.g., each of) an amino modifier C6, 5 thymines, a sequencing handle(e.g., R1), a 16 base pair barcode, a 12 base pair, UMI, and a 7 basepair ligation handle (SEQ ID NO: 8). FIG. 44 shows the ligation handleat the 3′ end of the acceptor oligonucleotide (e.g., acceptoroligonucleotide 2). In some embodiments, the ligation handle can becomplementary to a sequence in the splint oligonucleotide. In someembodiments, the splint oligonucleotide can have a sequencecomplementary to a second ligation handle that is present in anoligonucleotide that also includes the capture domain. In someembodiments, the capture domain can be any of the capture domainsdescribed herein.

In some embodiments, the splint oligonucleotide includes a sequence thatis complementary to an oligonucleotide (e.g., an acceptoroligonucleotide), or a portion thereof, and a sequence that iscomplementary to an oligonucleotide (e.g., a donor oligonucleotide)containing a capture domain, or a portion thereof (e.g., complementaryto the capture domain itself or to a second ligation handle). In someembodiments, the splint oligonucleotide includes a sequence that isperfectly complementary (e.g., is 100% complementary) to anoligonucleotide (e.g., an acceptor oligonucleotide), or a portionthereof, and/or a sequence that is perfectly complementary to anoligonucleotide containing a capture domain, or a portion thereof (e.g.,perfectly complementary to the capture domain itself or to a secondligation handle). In some embodiments, the splint oligonucleotideincludes a sequence that is not perfectly complementary (e.g., is not100% complementary) to an oligonucleotide (e.g., an acceptoroligonucleotide), or a portion thereof, and/or a sequence that is notperfectly complementary to an oligonucleotide containing a capturedomain, or a portion thereof (e.g., not perfectly complementary to thecapture domain itself or to a second ligation handle). In someembodiments, the splint oligonucleotide includes a sequence that is atleast 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementaryto an oligonucleotide (e.g., an acceptor oligonucleotide), or a portionthereof, and/or a sequence that is complementary to an oligonucleotidecontaining a capture domain, or a portion thereof (e.g., at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to thecapture domain itself or to a second ligation handle). In someembodiments, the splint oligonucleotide includes a sequence that isperfectly complementary (e.g., is 100% complementary) to anoligonucleotide (e.g., an acceptor oligonucleotide), or a portionthereof, but is not perfectly complementary to a sequence that iscomplementary to an oligonucleotide containing a capture domain, or aportion thereof (e.g., not perfectly complementary to the capture domainitself or to a second ligation handle). In some embodiments, the splintoligonucleotide includes a sequence that is not perfectly complementary(e.g., is not 100% complementary) to an oligonucleotide (e.g., anacceptor oligonucleotide), or a portion thereof, but is perfectlycomplementary to a sequence that is complementary to an oligonucleotidecontaining a capture domain, or a portion thereof (e.g., perfectlycomplementary to the capture domain itself or to a second ligationhandle). So long as the splint oligonucleotide is capable of hybridizingto an oligonucleotide (e.g., an acceptor oligonucleotide), or a portionthereof, and to a sequence that is complementary to an oligonucleotidecontaining a capture domain, or a portion thereof (e.g., to the capturedomain itself or to a second ligation handle), the splintoligonucleotide need not have a sequence that is perfectly complementaryto either the oligonucleotide (e.g., the acceptor oligonucleotide) or tothe an oligonucleotide containing a capture domain.

The splint oligonucleotide can be about 20 to about 80 nucleotides long.In some embodiments, the splint oligonucleotide can be about 30 to about70 nucleotides long. In some embodiments, the splint oligonucleotide canbe about 40 to about 60 nucleotides long. In some embodiments, thesplint oligonucleotide can be about 45 to about 55 nucleotides long.

In some embodiments, the spatial array is generated after ligatingcapture domains (e.g., poly(T) or gene specific capture domains) to theoligonucleotides (e.g., generating capture probes). The spatial arraycan be used with any of the spatial analysis methods described herein.For example, a biological sample can be provided to the generatedspatial array generated. In some embodiments, the biological sample ispermeabilized. In some embodiments, the biological sample ispermeabilized under conditions sufficient to allow one or more analytespresent in the biological sample to interact with the capture probes ofthe spatial array. After capture of analytes in the biological samplethe analytes can be analyzed (e.g., reverse transcribed, amplified,sequenced) by any of the variety of methods described herein.

Exemplary Embodiments

This disclosure relates to methods for building an array. In one aspect,provided herein are methods of generating an array for spatial analysisthat includes: (a) providing an array that includes a plurality of insitu synthesized oligonucleotides, wherein each of the in situsynthesized oligonucleotides comprise a spatial barcode, and wherein the3′ end of each of the in situ synthesized oligonucleotides are attachedto a solid support; (b) providing a plurality of primers complementaryto a portion of one or more in situ synthesized oligonucleotides; (c)extending the primers using the one or more in situ synthesizedoligonucleotides as a template, thereby generating a firstoligonucleotide; (d) extending the first oligonucleotide to produce a 3′end overhang; (e) ligating a splint oligonucleotide to the 5′ end of theone or more in situ synthesized oligonucleotides; and (f) ligating asecond oligonucleotide to the 3′ end of the first oligonucleotide, wherethe second oligonucleotide includes a sequence that is complementary toa portion of the splint oligonucleotide and a capture domain, therebygenerating a plurality of capture probes to be used in an array forspatial analysis. In some embodiments, the splint oligonucleotideincludes a sequence that is complementary to the 3′ end overhang of thefirst oligonucleotide. In some embodiments, the plurality of in situsynthesized oligonucleotides include a first constant sequence. In someembodiments, the plurality of in situ synthesized oligonucleotidesfurther comprise a second constant sequence. In some embodiments, theplurality of in situ synthesized oligonucleotides have a length ofbetween 1 and 30 nucleotides. In some embodiments, the secondoligonucleotide further includes a constant sequence, and where theconstant sequence is complementary to a portion of the splintoligonucleotide. In some embodiments, the second oligonucleotide furtherincludes a degenerate sequence. In some embodiments, the capture domainincludes a poly-T sequence. In some embodiments, the 3′ end overhang ofstep (d) is produced by an enzyme. In some embodiments, the enzyme is atemplate-independent polymerase. In some embodiments, thetemplate-independent polymerase is at least one of terminaldeoxynucleotidyl transferase (TdT) and poly A polymerase.

In one aspect, provided herein are methods for spatial analysis of abiological sample that includes: (a) providing an array produced by themethod of claim 1; (b) contacting a biological sample with the array;(c) permeabilizing the biological sample under conditions sufficient toallow a plurality of biological analytes in the biological sample tointeract with a plurality of capture probes on the array to generate aplurality of spatially barcoded biological analytes; and analyzing theplurality of spatially barcoded biological analytes, thereby determiningthe spatial location of the plurality of biological analytes.

Capture Probes

In some embodiments, the capture probe is a product of two or moreoligonucleotide sequences. In some embodiments, two or moreoligonucleotide sequences are ligated together. In some embodiments, thecapture probe includes a splint oligonucleotide. In some embodiments,the capture probe includes one or more constant sequences. In someembodiments, the capture probe is generated by having an enzyme addpolynucleotides at the end of an oligonucleotide sequence. In someembodiments, the capture probe includes a degenerate sequence. In someembodiments, the degenerate sequence functions as a unique molecularidentifier.

In Situ Synthesized Oligonucleotides

In some embodiments, the capture probe includes an in situ synthesizedoligonucleotide. In some embodiments, the in situ synthesizedoligonucleotide includes one or more constant sequences. In someembodiments, a constant sequence may be a cleavable sequence. In someembodiments, the in situ synthesized oligonucleotide includes a barcodesequence. In some embodiments, the in situ synthesized oligonucleotideincludes a variable barcode sequence. In some embodiments, the in situsynthesized oligo have a length of between 1 and 50 nucleotides (e.g.between 1 and 40, between 1 and 30, between 1 and 20, or between 1 and10 nucleotides). In some embodiments, the in situ synthesizedoligonucleotide is attached to an array. In some embodiments, the insitu synthesized oligonucleotide is attached to a feature. In someembodiments, the attachment is direct attachment. In some embodiments,the attachment is an indirect attachment.

Splint Oligonucleotides

The term “splint oligonucleotide”, as used herein, refers to anoligonucleotide that, when hybridized to other polynucleotides, acts asa “splint” to position the polynucleotides next to one another so thatthey can be ligated together. In some embodiments, the splintoligonucleotide is DNA. In some embodiments, the splint oligonucleotideis RNA. In some embodiments, the splint oligonucleotide comprises anucleotide sequence that is partially complimentary to nucleotidesequences from two or more different oligonucleotides. In someembodiments, the splint oligonucleotide assists in ligating a “donor”oligonucleotide and an “acceptor” oligonucleotide. In some embodiments,the splint oligonucleotide is7777 approximately 40 nucleotides inlength. In some embodiments, the splint oligonucleotide is between 10and 50 oligonucleotides in length. In some embodiments, the splintoligonucleotide is between 10 and 45, 10 and 40, 10 and 35, 10 and 30,10 and 25, or 10 and 20 oligonucleotides in length. In some embodiments,the splint oligonucleotide is between 15 and 50, 15 and 45, 15 and 40,15 and 35, 15 and 30, 15 and 30, or 15 and 25 nucleotides in length. Insome embodiments, an RNA ligase is used to ligate two nucleotidesequences together. In some embodiments, a DNA ligase is used to ligatetwo oligonucleotide sequences together. In some embodiments, the DNAligase is a T4 DNA ligase.

Degenerate Sequence

In some embodiments of any of the methods described herein, the captureprobes may include a degenerate sequence. In some embodiments, adegenerate sequence is a sequence in which some positions of anucleotide sequence contain a number of possible bases. In someembodiments of any of the methods described herein, a degeneratesequence can be a degenerate nucleotide sequence including about or atleast 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 45, or 50 nucleotides. In some embodiments, a nucleotidesequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 0, 10, 15, 20, 25, or moredegenerate positions within the nucleotide sequence. In some embodimentsof any of the methods described herein, the degenerate sequence is usedas a unique molecular identifier.

This disclosure relates to methods for preparing high-density spatiallybarcoded arrays and methods for spatial analysis (e.g., analysis of thespatial location(s) of one or more species of analytes in a biologicalsample) of analytes in a biological sample using a high-densityspatially barcoded arrays.

In one aspect, provided herein are methods for preparing a high-densityspatially barcoded array, comprising: (a) contacting an array comprisingone or more pluralities of capture probes with a hydrogel including oneor more pluralities of oligonucleotides; (b) attaching members of theone or more pluralities of capture probes to members of the one or morepluralities of oligonucleotides to form one or more pluralities ofcoupled capture probes; (c) reducing the size of the hydrogel includingthe one or more pluralities of coupled capture probes by removing water,forming a shrunken hydrogel including the one or more pluralities ofcoupled capture probes; thus generating a high-density spatiallybarcoded array.

In some embodiments, the one or more pluralities of capture probes areimmobilized. In some embodiments, the one or more pluralities ofimmobilized capture probes are partitioned. In some embodiments, the oneor more pluralities of immobilized capture probes are partitioned into aplurality of capture probe partitions. In some embodiments, apartitioned plurality of immobilized capture probes is different fromanother partitioned plurality of immobilized capture probes.

In some embodiments, one or more first pluralities of capture probescomprise a barcode sequence. In some embodiments, one or morepluralities of capture probes comprise a barcode sequence different fromthe barcode sequence of one or more other pluralities of capture probes.In some embodiments, one or more capture probe partitions comprise aplurality of immobilized capture probes including a barcode sequencedifferent from the barcode sequence of the plurality of immobilizedcapture probes of one or more other capture probe partitions.

In some embodiments, the one or more pluralities of oligonucleotides arepartitioned. In some embodiments, the one or more pluralities ofoligonucleotides are partitioned into a plurality of oligonucleotidepartitions. In some embodiments, one or more oligonucleotide partitionscomprise a plurality of oligonucleotides different from one or moreother oligonucleotide partitions. In some embodiments, one or morepluralities of oligonucleotides comprise a barcode sequence. In someembodiments, one or more pluralities of oligonucleotides comprise abarcode sequence different from the barcode sequence of one or morepluralities of oligonucleotides. In some embodiments, the barcodesequence of one or more pluralities of capture probes is different fromthe barcode sequence of one or more pluralities of oligonucleotides. Insome embodiments, one or more oligonucleotide partitions comprise aplurality of oligonucleotides including a barcode sequence differentfrom the barcode sequence of the plurality of oligonucleotides of one ormore oligonucleotide partitions.

In some embodiments, the reduction in the size of the hydrogel isisometric.

In some embodiments, the removal of water comprises a dehydratingsolvent. In some embodiments, the solvent comprises at least one of analcohol, a ketone, an aldehyde, acetonitrile, or a combination thereof.In some embodiments, the solvent comprises at least one of glycerol,ethanol, acetonitrile, acetone, 2-propanol, and a combination thereof.

In some embodiments, the removal of water comprises a salt. In someembodiments, the salt comprises an inorganic salt.

In some embodiments, the removal of water comprises an acid.

In some embodiments, the removal of water comprises a dehydratingprocess.

In some embodiments, the shrunken hydrogel is about 2 to about 10 foldsmaller in a linear dimension than the pre-shrunk hydrogel. In someembodiments, the shrunken hydrogel is at least 2-fold smaller in alinear dimension than the pre-shrunk hydrogel. In some embodiments, theshrunken hydrogel is at least 4-fold smaller in a linear dimension thanthe pre-shrunk hydrogel. In some embodiments, the shrunken hydrogel isat least 8-fold smaller in a linear dimension than the pre-shrunkhydrogel.

In some embodiments, the attaching comprises polymerase chain reaction.

In some embodiments, the hydrogel comprises a DNA polymerase, aplurality of primers, and a plurality of nucleotides.

In some embodiments, members of the plurality of capture probes arecross-linked to the hydrogel.

In some embodiments, members of the one or more pluralities of coupledcapture probes are amplified.

In some embodiments, the coupled capture probe is amplified prior to thereduction of the size of the hydrogel.

In some embodiments, the hydrogel is prepared from hydrogel subunitsincluding at least one of: acrylamide, bis-acrylamide, a poly(ethyleneglycol), a polyaliphatic polyurethane, a polyether polyurethane, apolyester polyurethane, a polyethylene copolymer, a polyamide, apolyvinyl alcohol, polypropylene glycol, polytetramethylene oxide,polyvinyl pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate),poly(hydroxyethyl methacrylate), collagen, hyaluronic acid, chitosan,dextran, agarose, gelatin, alginate, a protein polymer, methylcellulose,and a combination thereof.

In some embodiments, one or more capture probes comprise a spatialbarcode. In some embodiments, one or more capture probes comprise aunique molecular identifier. In some embodiments, one or morepluralities of capture probes comprise a spatial barcode, and a capturedomain.

Also provided herein are methods for spatially profiling a plurality ofanalytes in a biological sample, including: (a) providing a high-densityspatially barcoded array as described herein; (b) contacting thebiological sample with the high-density spatially barcoded array underconditions such that one or more analytes present in the biologicalsample are captured by one or more features of the array; and (c)determining the spatial profile of the captured analytes.

In some embodiments, the biological sample is a tissue sample. In someembodiments, the tissue sample is a fresh-frozen tissue sample. In someembodiments, the tissue sample comprises a tumor cell. In someembodiments, the tissue sample comprises a tissue section.

In some embodiments, the analytes in the biological sample are migratedto the bead array. In some embodiments, the migrating comprises passivemigration. In some embodiments, the migrating comprises activemigration.

In some embodiments, the one or more analytes comprises at least one ofRNA, DNA, protein, lipid, peptide, metabolite, small molecule, and acell labeling agent.

Methods for Preparing High-Density Spatially Barcoded Arrays

Provided herein are methods for preparing a high-density spatiallybarcoded array, e.g., a high-diversity and high-density spatiallybarcoded array. For example, methods for preparing a high-densityspatially barcoded array can include attaching capture probes tooligonucleotides in a hydrogel to form coupled capture probes andreducing the size of the hydrogel including the coupled capture probes.In some embodiments, a method for preparing a high-density spatiallybarcoded array, can include contacting an array including one or morepluralities of capture probes with a hydrogel including one or morepluralities of oligonucleotides; attaching members of the one or morepluralities of capture probes to members of the one or more pluralitiesof oligonucleotides to form one or more pluralities of coupled captureprobes; reducing the size of the hydrogel including the one or morepluralities of coupled capture probes by removing water, forming ashrunken hydrogel including the one or more pluralities of coupledcapture probes; thus generating a high-density spatially barcoded array.

In some embodiments, a capture probe is any capture probe as describedherein. In some embodiments, the one or more pluralities of captureprobes are immobilized.

The term “partition,” as used herein, may be a verb or a noun. When usedas a verb (e.g., “to partition,” or “partitioning”), the term generallyrefers to the fractionation (subdivision) of a species or sample (e.g.,a polynucleotide) such that one fraction (or subdivision) is sequesteredfrom another. Each sequestered section is referred to using the noun“partition.” In some embodiments, a partition maintains separation ofits own contents from the contents of other partitions. A partition maybe any suitable area in which one fraction of a sample is separated orsequestered from another. In some embodiments, a hydrogel can compriseone or more partitions of a species or sample (e.g., a polynucleotide),wherein the hydrogel matrix maintains the separation of the contents ofone partition from the contents of other partitions. In someembodiments, a partition has defined dimensions.

In some embodiments, the one or more pluralities of immobilized captureprobes are partitioned. In some embodiments, the one or more pluralitiesof immobilized capture probes are partitioned into a plurality ofcapture probe partitions. In some embodiments, a partitioned pluralityof immobilized capture probes is different from another partitionedplurality of immobilized capture probes.

In some embodiments, one or more first pluralities of capture probescomprise a barcode sequence. In some embodiments, one or morepluralities of capture probes comprise a barcode sequence different fromthe barcode sequence of one or more other pluralities of capture probes.In some embodiments, one or more capture probe partitions comprise aplurality of immobilized capture probes including a barcode sequencedifferent from the barcode sequence of the plurality of immobilizedcapture probes of one or more other capture probe partitions.

In some embodiments, the one or more pluralities of oligonucleotides arepartitioned. In some embodiments, the one or more pluralities ofoligonucleotides are partitioned into a plurality of oligonucleotidepartitions. In some embodiments, one or more oligonucleotide partitionscomprise a plurality of oligonucleotides different from one or moreother oligonucleotide partitions. In some embodiments, one or morepluralities of oligonucleotides comprise a barcode sequence. In someembodiments, one or more pluralities of oligonucleotides comprise abarcode sequence different from the barcode sequence of one or morepluralities of oligonucleotides. In some embodiments, the barcodesequence of one or more pluralities of capture probes is different fromthe barcode sequence of one or more pluralities of oligonucleotides. Insome embodiments, one or more oligonucleotide partitions comprise aplurality of oligonucleotides including a barcode sequence differentfrom the barcode sequence of the plurality of oligonucleotides of one ormore oligonucleotide partitions.

In some embodiments, each capture probe comprises a spatial barcode. Insome embodiments, each capture probe from the plurality of captureprobes comprises a unique molecular identifier. In some embodiments,each capture probe comprises a cleavage domain, a spatial barcode, aunique molecular identifier, and a capture domain.

A “coupled capture probe” as used herein refers to a capture probe thatis attached to an oligonucleotide (e.g., an oligonucleotide in ahydrogel). For example, one or more domains of a capture probe can beattached to one or more domains of an oligonucleotide (e.g., anoligonucleotide in a hydrogel). “Attaching” can refer to any method thatbinds a capture probe to an oligonucleotide (e.g., an oligonucleotide ina hydrogel). For example, an oligonucleotide can be ligated to an end ofa capture probe. As another example, PCR can be used to attach one ormore domains of a capture probe to one or more domains of anoligonucleotide (e.g., an oligonucleotide in a hydrogel).

In some embodiments, members of the one or more pluralities of captureprobes comprise a nucleic acid, e.g., an oligonucleotide. In someembodiments, attaching members of the one or more pluralities of captureprobes to members of the one or more pluralities of oligonucleotides toform one or more pluralities of coupled capture probes comprisespolymerase chain reaction (PCR). In some embodiments, the hydrogelincludes PCR reagents such as a DNA polymerase, a plurality of primers,and a plurality of nucleotides. In some embodiments, members of theplurality of features are amplified (see, e.g., Mitra and Church,Nucleic Acids Res. 1999 Dec 15;27(24):e34, which is incorporated byreference herein in its entirety).

In some embodiments, chemical ligation can be used to ligate two or moreoligonucleotides (e.g., a capture probe and an oligonucleotide from ahydrogel as described herein). In some embodiments, chemical ligationinvolves the use of condensing reagents. In some embodiments, condensingreagents are utilized to activate a phosphate group. In someembodiments, condensing reagents may be one or more of 1- 25ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI), cyanogen bromide,imidazole derivatives, and 1-hydroxybenzotriazole (HOAt). In someembodiments, functional group pairs selected from one or more of anucleophilic group and an electrophilic group, or an alkyne and an azidegroup are used for chemical ligation. In some embodiments, chemicalligation of two or more oligonucleotides requires a template strand thatis complementary to the oligonucleotides to be ligated. In someembodiments, the chemical ligation process is similar to oligonucleotidesynthesis.

In some embodiments, not all of the domains of a capture probe areattached to an oligonucleotide to form a coupled capture probe. Forexample, a coupled capture probe may comprise 1, 2, 3, 4, or 5 domainsof the original capture probe. In some embodiments, all the domains ofthe capture probe are attached to the oligonucleotide. For example, thecoupled capture probe comprises all the domains of the original captureprobe.

In some embodiments, members of the one or more pluralities of captureprobes are cross-linked to a hydrogel.

As used herein “shrinking” or “reducing the size of” a hydrogel refersto any process causing the hydrogel to physically contract and/or thesize of the hydrogel to decrease in volume. For example, the scaffold ofthe gel may shrink or “implode” upon solvent removal (see, e.g., Longand Williams. Science. 2018; 362(6420):1244-1245, and Oran et al.Science. 2018; 362(6420):1281-1285; each of which is incorporated hereinby reference in its entirety). As another example, the process to shrinkor reduce the size of a hydrogel may be one that removes water (i.e., adehydrating process) from the hydrogel. There are many methods known toone of skill in the art for shrinking or reducing the size of ahydrogel. Non-limiting examples of a method to shrink or reduce the sizeof a hydrogel include exposing the hydrogel to one or more of: adehydrating solvent, a salt, heat, a vacuum, lyophilization,desiccation, filtration, and air-drying.

In some embodiments, the removal of water comprises a dehydratingsolvent. Non-limiting examples of a dehydrating solvent include a ketone(such as methyl ethyl ketone (MEK)), isopropanol (IPA), acetone,1-butanol, methanol (MeOH), dimethyl sulfoxide (DMSO), glycerol,propylene glycol, ethylene glycol, ethanol, (k) 1,4-dioxane, propylenecarbonate, furfuryl alcohol, N,N-dimethylformamide (DMF), acetonitrile,and an aldehyde (such as formaldehyde or glutaraldehyde). In someembodiments, the solvent comprises at least one of an alcohol, a ketone,an aldehyde, acetonitrile, and a combination thereof. In someembodiments, the solvent comprises a solvent selected from the groupconsisting of: glycerol, ethanol, acetonitrile, acetone, 2-propanol, anda combination thereof.

In some embodiments, the removal of water comprises a salt. In someembodiments, the salt comprises an inorganic salt. Non-limiting examplesof salts that may be used to shrink a hydrogel include inorganic saltssuch as aluminum, ammonium, barium, beryllium, calcium, cesium, lithium,magnesium, potassium, rubidium, sodium, and strontium salts. Furthernon-limiting examples of inorganic salts include sodium chloride,potassium chloride, lithium chloride, cesium chloride, sodium fluoride,sodium bromide, sodium iodide, sodium nitrite, potassium sulfate,potassium nitrate, potassium carbonate, potassium bicarbonate, sodiumsulfate, sodium nitrate, sodium carbonate, sodium bicarbonate, calciumsulfate, copper oxychloride, calcium chloride, calcium carbonate,calcium bicarbonate, magnesium sulfate, magnesium nitrate, magnesiumchloride, magnesium carbonate, magnesium bicarbonate, ammonium sulfate,ammonium chloride, ammonium nitrate, ammonium carbonate, ammoniumbicarbonate, trisodium phosphate, tripotassium phosphate, calciumphosphate, copper(II) sulfate, sodium sulfide, potassium sulfide,calcium sulfide, potassium permanganate, iron(II) chloride, iron(III)chloride, iron (2+) sulfate, iron(III) sulfate, iron(II) nitrate,iron(III) nitrate, manganese(II) chloride, manganese(III) chloride,manganese(II) sulfate, manganese(II) nitrate, zinc chloride, zincnitrate, zinc sulfate, ammonium orthomolybdate, monopotassium phosphate,nickel(II) sulfate, nickel(II) nitrate, sodium metavanadate, sodiumparavanadate, potassium dichromate, ammonium dichromate, antipyonin,ammonium nitrite, potassium fluoride, sodium fluoride, ammoniumfluoride, calcium fluoride, chrome alum, potassium alum, potassiumiodide, sodium hypochlorite, tin(II) sulfate, tin(II) nitrate, goldselenite, dicesium chromate, potassium perchlorate, calcium perchlorate,aluminium sulphate, lead(II) bisulfate, barium phosphate, bariumhydrogen orthophosphate, barium dihydrogen phosphate, silver dichromate,potassium bromate, sodium bromate, sodium iodate, sodium silicate,diammonium phosphate, ammonium phosphate, ammonium dihydrogen phosphate,chromium orthophosphate, copper(II) chloride, copper(I) chloride, sodiumtetrametaphosphate, potassium heptafluoroniobate, zinc phosphate, sodiumsulfite, copper(I) nitrate, copper(II) nitrate, potassium silicate,copper(II) carbonate basic, copper(II) carbonate salts of acrylic acidand sulfopropyl acrylate.

In some embodiments, the removal of water comprises an acid. In someembodiments, the hydrogel is exposed to a different pH environment. Forexample, the hydrogel can be exposed to an acidic pH or a basic pH. Insome embodiments, the hydrogel is exposed to a pH of less than about6.5, e.g., a pH of about 6, about 5.5, about 5, about 4.5, about 4,about 3.5, about 3, about 2.5, about 2, about 1.5, or about 1. In someembodiments, the hydrogel is exposed to a pH of greater than about 7.5,e.g., a pH of about 8, about 8.5, about 9, about 9.5, about 10, about10.5, about 11, about 11.5, 10 about 12, about 12.5, about 13, about13.5, or about 14.

In some embodiments, the removal of water comprises a dehydratingprocess such as heat, a vacuum, lyophilization, desiccation, filtration,and air-drying. In some embodiments, the hydrogel is exposed to anincreased temperature, e.g., a higher temperature than about roomtemperature. For example, a temperature of about 37° C., 38.15° C., 39°C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48°C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57°C., 58° C., 59° C. 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66°C., 67° C., 68° C., 69° C., or 70° C., or higher. As another example,the hydrogel is exposed to an increase in temperature from about 37° C.,38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C.,47° C., 48° C., or 49° C. to about 50° C., 51.20° C., 52° C., 53° C.,54° C., 55° C., 56° C., 57° C., 58° C., 59° C. 60° C., 61° C., 62° C.,63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., or 70° C., orhigher).

In some embodiments, more than one method to shrink or reduce the sizeof a hydrogel can be used. For example, a hydrogel can be exposed to anacid and subsequently dehydrated by another method such as heat, avacuum, lyophilization, desiccation, filtration, or a combinationthereof.

In some embodiments, the hydrogel can further comprise a cross-linkingagent. The cross linking agent may help stabilize the hydrogel and/ormodulate the extent the hydrogel shrinks. Non-limiting examples of across-linking agent include disuccinimidyl suberate (DSS),dimethylsuberimidate (DMS), formalin, dimethyladipimidate (DMA),dithio-bis(-succinimidyl propionate) (DSP), disuccinimidyl tartrate(DST), and ethylene glycol bis(succinimidyl succinate) (EGS). In someembodiments, the shrunken hydrogel is at least about 2-fold smaller in alinear dimension (e.g., along one axis) than the pre-shrunk hydrogel.For example, at least about 2.5, about 3, about 4, about 5, about 6,about 7, about 8, about 9, about 10, or more fold smaller in a lineardimension than the pre-shrunk hydrogel. In some embodiments, a shrunkenhydrogel is about 2 to about 10 fold smaller in a linear dimension thanthe pre-shrunk hydrogel. For example, about 2 to about 3, about 2 toabout 4, about 2 to about 5, about 2 to about 6, about 2 to about 7,about 2 to about 8, about 2 to about 9, about 9 to about 10, about 8 toabout 10, about 7 to about 10, about 6 to about 10, about 5 to about 10,about 4 to about 10, or about 3 to about 10. In some embodiments, ashrunken hydrogel is about 2 to about 4, about 3 to about 5, about 4 toabout 6, about 5 to about 7, about 6 to about 8 fold smaller in a lineardimension than the pre-shrunk hydrogel. In some embodiments, a shrunkenhydrogel is about 2, about 3, about 4, about 5, about 6, about 7, about8, about 9, or about 10 fold smaller in a linear dimension than thepre-shrunk hydrogel.

In some embodiments, the size of the hydrogel is reduced along more thanone axes, e.g., along 2 or 3 axes. In some embodiments, each axisintersects each other axis at 90 degrees. In some embodiments, the sizeof the hydrogel along the first axis is about 20 2 to about 10 or morefold smaller than the pre-shrunk hydrogel, e.g., about 2, about 3, about4, about 5, about 6, about 7, about 8, about 9, about 10 or more foldsmaller than the pre-shrunk hydrogel. In some embodiments, the size ofthe hydrogel along the second axis is about 2 to about 10 or more foldsmaller than the pre-shrunk hydrogel, e.g., about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10 or more foldsmaller than the pre-shrunk hydrogel. In some embodiments, the size ofthe hydrogel along the third axis is about 2 to about 10 or more foldsmaller than the preshrunk hydrogel, e.g., about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10 or more foldsmaller than the pre-shrunk hydrogel. In some embodiments, the reductionin the size of the hydrogel is isometric.

In some embodiments, a hydrogel is prepared from hydrogel subunitsincluding at least one of: acrylamide, bis-acrylamide, a poly(ethyleneglycol), a polyaliphatic polyurethane, a polyether polyurethane, apolyester polyurethane, a polyethylene copolymer, a polyamide, apolyvinyl alcohol, polypropylene glycol, polytetramethylene oxide,polyvinyl pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate),poly(hydroxyethyl methacrylate), collagen, hyaluronic acid, chitosan,dextran, agarose, gelatin, alginate, a protein polymer, methylcellulose,and a combination thereof.

In some embodiments, the size of a hydrogel including coupled captureprobes is reduced, thus increasing the density of the coupled captureprobe. In some embodiments, the volume of a partition of members of theplurality of capture probes in a hydrogel is reduced when the size ofthe hydrogel is reduced.

Methods for Spatial Analysis of Analytes Using a Barcoded BiologicalSample

In some embodiments, a high-density spatially barcoded array (e.g., ahigh density spatially barcoded array as described herein) can be usedfor spatial analysis of analytes in the biological sample. For example,methods for spatially profiling a plurality of analytes in a biologicalsample can include providing a high-density spatially barcoded array asdescribed herein; contacting the biological sample with the high-densityspatially barcoded array under conditions such that one or more analytespresent in the biological sample are captured by one or more captureprobes of the array; and determining the spatial profile of the capturedanalytes.

This disclosure relates to methods for building an array. In someembodiments, methods of generating an array comprise: (a) providing anarray including a plurality of features immobilized on a solidsubstrate, wherein each feature comprises a plurality of in situsynthesized oligonucleotides, and wherein each in situ synthesizedoligonucleotide comprises a first barcode; (b) selecting one or more insitu synthesized oligonucleotides from the plurality of in situsynthesized oligonucleotides; (c) delivering an oligonucleotide to theone or more selected in situ synthesized oligonucleotides, wherein theoligonucleotide comprises a second barcode and a capture domain; and (d)attaching the oligonucleotide to the one or more selected in situsynthesized oligonucleotides, thereby generating one or more captureprobes including the first barcode and the second barcode, therebygenerating an array.

In some embodiments, each feature of the plurality of features isassociated with one of a number of different first barcodes. In someembodiments, each oligonucleotide is associated with one of a number ofdifferent second barcodes.

In some embodiments, step (d) comprises ligating the oligonucleotide tothe one or more selected in situ synthesized oligonucleotides. In someembodiments, the method comprises ligating the oligonucleotide to theone or more selected in situ synthesized oligonucleotides using aligase. In some embodiments, the oligonucleotide is attached to the oneor more selected in situ synthesized oligonucleotides by a chemicalcoupling reaction. In some embodiments, each of the in situ synthesizedoligonucleotide comprises a 3′ end that is masked by a protective group.In some embodiments, the method comprises removing the protective groupfrom the one or more selected in situ synthesized oligonucleotides priorto delivering an oligonucleotide to the one or more selected in situsynthesized oligonucleotides. In some embodiments, the protective groupis a photocleavable protective group. In some embodiments, theprotective group is removed by exposing the one or more selected in situsynthesized oligonucleotides to light. In some embodiments, theprotective group coupled to the one or more selected in situ synthesizedoligonucleotides is removed by applying a photomask.

In some embodiments, the selected in situ synthesized oligonucleotidesare separated from the remaining in situ synthesized oligonucleotides byone or more physical barriers, wherein the one or more physical barriersform one or more chambers, and wherein the oligonucleotide is deliveredto the one or more chambers including the one or more selected in situsynthesized oligonucleotides. In some embodiments, the one or morephysical barriers include a hydrophobic surface. In some embodiments,the one or more physical barriers include one or more chamber wallshaving a tapered end. In some embodiments, the oligonucleotide comprisesa unique molecular identifier. In some embodiments, the oligonucleotidecomprises a 3′ end that is masked. In some embodiments, a free 3′ -OHgroup on the capture probes is created.

In some embodiments, methods of generating an array comprise: (a)providing an array including two or more partitions, wherein eachpartition comprises a plurality of features immobilized on a substrate,wherein each feature comprises a plurality of in situ synthesizedoligonucleotides, and wherein each in situ synthesized oligonucleotidecomprises a first barcode; (b) delivering an oligonucleotide to a firstpartition, wherein the oligonucleotide comprises a second barcode and acapture domain; and (c) attaching the oligonucleotide to one or more insitu synthesized oligonucleotides in the first partition, therebygenerating one or more capture probes including the first barcode andthe second barcode in the first partition, thereby generating an array.

In some embodiments, methods provided herein further comprise: (d)delivering a second oligonucleotide to a second partition, wherein thesecond oligonucleotide includes a third barcode; and (e) attaching thesecond oligonucleotide to one or more in situ synthesizedoligonucleotides in the second partition, thereby generating captureprobes including the first barcode and the third barcode in the secondpartition.

In some embodiments, the two or more partitions are separated by one ormore physical barriers. In some embodiments, the one or more physicalbarriers are printed on the solid substrate. In some embodiments, theone or more physical barriers form one or more chambers comprising oneor more chamber walls. In some embodiments, the one or more chamberwalls comprise a hydrophobic surface. In some embodiments, the one ormore chamber walls comprise a tapered end in contact with the solidsubstrate. In some embodiments, the second nucleotide is delivered usinga microfluidic device.

Chemical Ligation of Oligonucleotides

In some embodiments, chemical ligation can be used to ligate two or moreoligonucleotides. In some embodiments, chemical ligation involves theuse of condensing reagents. In some embodiments, condensing reagents areutilized to activate a phosphate group. In some embodiments, condensingreagents may be one or more of 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), cyanogen bromide, imidazole derivatives, and1-hydroxybenzotriazole (HOAt). In some embodiments, functional grouppairs selected from one or more of a nucleophilic group and anelectrophilic group, or an alkyne and an azide group are used forchemical ligation. In some embodiments, chemical ligation of two or moreoligonucleotides requires a template strand that is complementary to theoligonucleotides to be ligated. In some embodiments, the chemicalligation process is similar to oligonucleotide synthesis.

Photocleavable Protective Group

In some embodiments, a photocleavable protective group (e.g. anyphotocleavable, photoremovable, photoreleasable, photoactivatable, orphotosensitive protective group described herein or known in the art)may be coupled to one or both ends of an in situ synthesizedoligonucleotide, a tagging probe (e.g. any of the tagging probesdescribed herein), or a capture probe (e.g. any of the capture probedescribed herein). In some embodiments, a photocleavable protectivegroup is a chemical modification to a molecule that can be removed withlight. In some embodiments, the photocleavable protective group (PPG) isat least one of a nitrobenzyl-based PPG, a carbonyl-based PPG, or abenzyl-based PPG. In some embodiments, the photocleavable protectivegroup is removed from one or more tagging probes by exposing the one ormore tagging probes to a light.

The present disclosure provides sample preparation techniques that allowthe enrichment of target analytes from a library, based on a barcodecorresponding to a set spatial region of interest. Procedures forbarcoding and isolating analytes from single cells are herein discussed.Enrichment of select target spatial barcodes corresponding to analytesof interest is more efficient, as not all analytes from a library aresubject to downstream processes. Thus enrichment of analytes based onspatial barcodes may allow downstream processes such as sequencing to bemore efficient or provide higher quality data from the desired analytes.

In some embodiments provided is a method for enriching a population ofcapture probes comprising: (a) providing a plurality of capture probeswherein:

(i) members of the plurality of capture probes comprise a plurality ofdifferent spatial barcodes, and (ii) each member of the population ofcapture probes to be enriched comprises a target spatial barcode; and(b) enriching the population of capture probes comprising the targetspatial barcode.

In some embodiments provided is a method for spatial analysis of atissue comprising: (a) providing a solid substrate comprising aplurality of capture probes, wherein a subset of capture probes isattached at a predetermined position on the solid substrate, and whereineach capture probe of the subset comprises an identical spatial barcode;(b) contacting a tissue sample with the solid substrate such that thesubset of capture probes interacts with a plurality of analytes from thetissue sample to generate a plurality of spatially barcoded analytemolecules, wherein a member of the plurality of spatially barcodedanalyte molecules comprises the identical spatial barcode or acomplement thereof; (c) identifying a spatial region of interest withinthe tissue sample, wherein the spatial region of interest comprises theplurality of spatially barcoded analyte molecules; and (d) enriching theplurality of spatially barcoded analyte molecules for spatial analysisof the tissue sample.

In some embodiments, the solid substrate further comprises an additionalsubset of capture probes attached at a predetermined position on thesolid substrate, wherein a member of the additional subset of captureprobes comprises a second identical spatial barcode that is differentfrom the spatial barcode.

In some embodiments, the solid substrate further comprises an additionalsubset of capture probes attached at a predetermined position on thesolid substrate, wherein a member of the additional subset of captureprobes comprises a third identical spatial barcode that is differentfrom the previously used spatial barcodes.

In some embodiments, the capture probe comprises an analyte capturedomain.

In some embodiments, the analyte comprises at least one of RNA, DNA, anda cell labeling agent.

In some embodiments, the cell labelling agent comprises an antigenbinding molecule.

In some embodiments, the cell labelling agent comprises a regionconfigured to couple to the capture domain of the capture probe.

In some embodiments, the spatial region of interest is identified byperforming shallow sequencing of at least a subset of the plurality ofspatially barcoded analyte molecules, and identifying a target spatialbarcode.

In some embodiments, the spatial region of interest is identified by atleast one of immunostaining, immunohistochemistry, microscopy, visualinspection, and combinations thereof.

In some embodiments, the method further comprises performing a nucleicacid extension reaction using (i) the plurality of spatially barcodedanalyte molecules and (ii) a primer comprising a sequence specific forthe identical spatial barcode or a complement thereof to generate anenriched plurality of spatially barcoded analyte molecules comprisingthe spatial barcode or a complement thereof.

In some embodiments, the analyte is amplified by performing a polymerasechain reaction (PCR).

In some embodiments, the method further comprises enriching a populationof capture probes present in a sub-spatial region of interest: (a)wherein the sub-spatial region of interest is located within the spatialregion of interest, wherein the sub-spatial region of interest comprisesa sub-plurality of capture probes, and wherein: (i) members of thesub-plurality of capture probes comprise identical sub-spatial barcodes,(ii) members of the sub-plurality of capture probes to be enrichedcomprise a second target spatial barcode sequence; and (iii) the secondtarget spatial barcode sequence is associated with the sub-spatialregion of interest; and (b) enriching the population of capture probescomprising the second target spatial barcode sequence.

In some embodiments, the method further comprises enriching a populationof capture probes present in a sub-sub-spatial region of interest: (a)wherein the sub-sub-spatial region of interest is located within thesub-spatial region of interest, wherein the sub-sub-spatial region ofinterest comprises a third plurality of capture probes, and wherein: (i)members of the third plurality of capture probes comprise a thirdplurality of spatially barcoded sequences, (ii) members of the thirdplurality of capture probes to be enriched comprise a third targetspatial barcode sequence; and (iii) the third target spatial barcodesequence is associated with the sub-sub-spatial region of interest; and(b) enriching the population of capture probes comprising the thirdtarget spatial barcode sequence.

In some embodiments, the subset of spatial barcodes comprise one uniquebarcode.

In some embodiments, a spatial barcode sequence of the subset of spatialbarcodes identifies an analyte derived from a single cell.

In some embodiments, the plurality of spatially barcoded analytemolecules corresponds to one or more mRNAs in the tissue sample.

In some embodiments, the plurality of spatially barcoded analytemolecules corresponds to one or more proteins in the tissue sample.

In some embodiments, the plurality of spatially barcoded analytemolecules corresponds to one or more antibodies in the tissue sample.

In some embodiments, the plurality of spatially barcoded analytemolecules corresponds to one or more T-cell receptors in the tissuesample.

In some embodiments, the plurality of spatially barcoded analytemolecules corresponds to one or more regions of genomic DNA in thetissue sample.

In some embodiments, the plurality of spatially barcoded analytemolecules corresponds to one or more regions of chromatin in the tissuesample.

In some embodiments, the plurality of spatially barcoded analytemolecules corresponds to a clustered regularly interspaced shortpalindromic repeat (CRISPR) ribonucleic acid (crRNA) or a single guideribonucleic acid (sgRNA) in the tissue sample. In some embodiments, theplurality of spatially barcoded analyte molecules corresponds to amicrobiome or a portion thereof.

Shallow Sequencing

As used herein, the coverage or depth of sequencing is the number ofunique reads that include a given nucleotide in a reconstructedsequence. As used herein, shallow sequencing is a small or low number ofunique reads of an identified analyte. In some embodiments, shallowsequencing is performed to identify a spatial region of interest. Insome embodiments, shallow sequencing is performed on an entire tissuesample. In some embodiments, shallow sequencing is performed on anidentified region of a tissue sample. In some embodiments, shallowsequencing is performed after identifying and/or selecting a region of atissue sample by some other method.

Spatial Analyte Capture

As used herein, spatial analyte capture may utilize nucleic acid probescomprising spatial barcode sequences, which are immobilized on a solidsubstrate to generate an array. The spatial barcode sequences correspondto the location of the capture probes on the array. In some embodiments,biological samples, such as tissue sections, are placed onto the arrayand the DNA is processed, e.g. fragmented and provided with a bindingdomain that hybridizes to the probes on the array. In some embodiments,DNA polymerase extension or ligation reaction is performed in the tissuesection on the array. In some embodiments, the probes to which theprocessed DNA in the tissue section binds, are extended using the boundDNA as a template to obtain complementary strands of DNA, which istherefore immobilized on the surface of the array. In some embodiments,probes to which the processed DNA in the tissue section binds, templatethe ligation of the bound DNA to a second strand of the probe or thestrand of the DNA hybridized to the probe may template the ligation ofthe other strand of DNA to the probe, which is therefore immobilized onthe surface of the array. In some embodiments, the spatial barcodesequences in the probes, the extended probes carry information about theposition of the “captured” DNA in the biological specimen, e.g. tissuesection. In some embodiments, the extended probes may be released fromthe surface of the array and analyzed, e.g. sequenced, which results ina genomic DNA “library” with exact positional information. In someembodiments, the sequence data can then be matched to a position in thebiological specimen, e.g. tissue section. For instance, the tissuesection may be visualized or imaged, e.g. stained and photographed,before or after the DNA synthesis or ligation step to facilitate thecorrelation of the positional tag in the DNA molecule with a positionwithin the tissue section and the sequence data may be overlaid on animage of the tissue section, e.g. using a computer, to display thedistribution pattern of any gene of interest across the tissue section.The visualization step is not essential as the tagged DNA molecule maybe correlated with a position in the tissue sample using other means,e.g. the unique profile of the molecules captured with the samepositional tag, i.e. at the same on the array, may enable the moleculeto be correlated to a position within the tissue sample based on thecharacteristics of cells or areas of cells within the tissue sample.Other analytes may be captured by this method, including but not limitedto RNA, specific sub-types of RNA, proteins, antibodies, genomic DNA,T-cell receptors, small molecules, or other material.

Target Barcode Sequence

In some embodiments, a target barcode sequence is a barcode sequencecorresponding to a spatial region of interest. In some embodiments, atarget barcode sequence is or includes a spatial barcode. In someembodiments, a target barcode sequence is identified by identifying aspatial region of interest and identifying the barcode associated withthe spatial region of interest on the array. In some embodiments, atarget barcode sequence is identified by performing shallow sequencing,and selecting a barcode based on the captured analytes. In someembodiments, a target barcode sequence is identified by aco-localization of a target barcode sequence with one or more additionalbarcode sequences. In some embodiments, a target barcode sequence isidentified manually. In some embodiments, a target barcode sequence isidentified using a computer. In some embodiments, a target barcodesequence is identified algorithmically. In some embodiments, the targetbarcode sequence corresponds to a plurality of spatial barcode sequencesthat identify a region, a sub-region, a sub-sub-region, or furtherspatial demarcation. In some embodiments, a target barcode sequence is aunique molecular identifier, which identifies one capture probe. In someembodiments, a target barcode sequence is used to identify andcharacterize captured analytes at single-cell resolution. In someembodiments, the identifying can comprise sequencing a plurality ofcapture probes to generate a plurality of sequencing reads, andanalyzing the plurality of sequencing reads to identify the at least onetarget barcode sequence. In some embodiments, each of the plurality ofcapture probes can further comprise one or more functional sequencesselected from the group consisting of a sequencing primer sequence and asequence configured to attach to a flow cell of a sequencer. In someembodiments, the plurality of capture probes can comprise, from 5′ to3′, a first functional domain (e.g., adapter sequence), a sequencederived from a template nucleic acid, and a second adapter sequence. Thefirst adapter sequence may comprise a first sequence configured toattach to a flow cell of a sequencer, a first sequencing primersequence, and a spatial barcode sequence. The second adapter sequencemay comprise a second sequencing primer sequence and a second sequenceconfigured to attach to a flow cell of a sequencer. The second adaptersequence can further comprise an index sequence.

Enriching Barcoded Nucleic Acids

In an aspect, the present disclosure provides methods of enriching asample for target barcoded nucleic acids. The methods may compriseproviding a plurality of capture probes comprising a plurality ofdifferent spatial barcode sequences, identifying at least one targetbarcode sequence from the plurality of different barcode sequences, andenriching the analyte detected (e.g., specifically bound to the captureprobe) by the capture probes comprising the at least one target barcodesequence. Enriching may be performed by purifying capture probescomprising the at least one target barcode from a plurality of captureprobes. Enriching may be performed by amplifying (i.e. increasing thenumbers of) capture probes comprising the at least one target barcodefrom a plurality of capture probes. The amplification may includeisothermal amplification. Enriching may be performed by both purifyingand amplifying the capture probe comprising the at least one targetbarcode from a plurality of capture probes.

Spatial barcodes can be included in capture probes. For example, thecapture probes can be co-partitioned into a partition with spatialbarcodes to facilitate the barcoding reaction for spatial analysis. Thespatial barcodes may be attached to a bead (e.g., in the context of acapture probe). The bead may be a gel bead. In some embodiments, thespatial barcodes are releasably attached to the bead or gel bead asdescribed elsewhere herein (e.g. via a cleavage domain). The pluralityof capture probes may be barcoded as described elsewhere herein.

In some embodiments, methods provided herein may comprise enrichingnucleic acids comprising the at least one target barcode sequence. Theenriching may comprise performing a nucleic acid extension reactionusing a capture probe comprising the at least one target barcodesequence and a primer comprising a sequence specific for the at leastone target barcode sequence to generate an enriched plurality of captureprobes comprising the at least one target barcode sequence. The nucleicacid extension reaction may be a polymerase chain reaction (PCR). Thenucleic acid extension reaction may comprise a polymerase. The nucleicacid extension reaction may result in amplification of the nucleic acid.

In some embodiments, methods provided herein may further compriseperforming an additional PCR on the enriched plurality of capture probescomprising the at least one target barcode sequence. The additional PCRmay comprise use of an additional primer comprising a sequence specificfor the enriched plurality of capture probes comprising the at least onetarget barcode sequence. In some embodiments, the additional primer maycomprise a sequence specific for the spatial barcode sequence of theenriched plurality of capture probes comprising the at least one targetbarcode sequence. In some embodiments, the additional primer maycomprise a sequence substantially specific for the spatial barcodesequence of the enriched plurality of capture probes comprising the atleast one target barcode sequence. For example, the additional primermay comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%or more sequence complementarity or sequence identity to the spatialbarcode sequence of the enriched plurality of capture probes comprisingthe at least one target barcode sequence. In some embodiments, theadditional primer may comprise a sequence specific for a 5′ region of aspatial barcode sequence of the enriched plurality of capture probescomprising the at least one target barcode sequence. In some cases, theadditional primer can comprise a sequence that has no (or low) sequencecomplementarity or identity to the spatial barcode sequence of theenriched plurality of capture probes comprising the at least one targetbarcode sequence.

In some embodiments, the additional primer may comprise one or morefunctional sequences that facilitate sequencing of the enrichedplurality of capture probes. The one or more functional sequences maycomprise a sequencing primer sequence. The one or more functionalsequences may comprise a sequence configured to attach to a flow cell ofa sequencer.

In some embodiments, methods provided herein may further comprise,subsequent to the nucleic acid extension reaction, performing a sizeselection process to remove unincorporated primers. The size selectionprocess may comprise using a gel or gel-like polymer. The size selectionprocess may use gel electrophoresis. In some cases, the size selectionprocess may comprise using a solid support. The solid support may be anarray.

In some embodiments, the enriched plurality of nucleic acids maycomprise an affinity group. The affinity group may be incorporated tothe enriched plurality of the nucleic acid via a polymerase extensionreaction with a primer comprising the affinity group. The affinity groupmay be incorporated via enzymatic reaction with a nucleotide and theaffinity group. The affinity group may be attached covalently ornon-covalently. The method may further comprise coupling the enrichedplurality of capture probes comprising the affinity group to a solidsupport specific for the affinity group. The solid support may be anarray. The solid support may be a polymer matrix. The solid support maybe a glass slide. The solid support may be a resin. In some embodiments,the solid support may comprise an antibody or antibody fragment. In someembodiments, the solid support comprises avidin or streptavidin and theaffinity group comprises (or is) biotin. In some embodiments, the solidsupport comprises maltose and the affinity group comprises (or is)maltose binding protein. In some embodiments, the solid supportcomprises maltose-binding protein and the affinity group comprises (oris) maltose.

In some embodiments, prior to the nucleic acid extension reaction, afirst capture probe complementary to a first portion of the at least onetarget barcode sequence and a second capture probe complementary to asecond portion of the at least one target barcode sequence may behybridized. The first capture probe can be ligated to the second captureprobe to generate the primer comprising the sequence specific for the atleast one target barcode sequence. The ligating may comprise using a DNAligase.

In some embodiments, the enriching can result in at least 10-foldenrichment of capture probes comprising the at least one target barcodesequence. In some embodiments, the enriching can result in at least20-fold enrichment of capture probes comprising the at least one targetbarcode sequence. The enrichment of nucleic acids may be observed by anincrease in the percent of nucleic acids comprising the at least onetarget barcode sequence in the enriched plurality of capture probescompared to the percent amount of nucleic acids comprising the at leastone target barcode sequence in the plurality of capture probes. Theenriching may result in at least 50-fold enrichment of capture probescomprising the at least one target barcode sequence. The enriching mayresult in at least 100-fold enrichment of capture probes comprising theat least one target barcode sequence. The enriching may result in atleast 200-fold enrichment of capture probes comprising the at least onetarget barcode sequence. The enriching may results in at least 500-foldenrichment of capture probes comprising the at least one target barcodesequence. The enriching may result in at least 1000-fold enrichment ofcapture probes comprising the at least one target barcode sequence.

Primers are used for multiple reactions described elsewhere herein. Theprimers may comprise a specific length of nucleotides. For example, theprimers may be at least about 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22,24, 25, 26, 28, 30, 40, 50 or more nucleotides long. Alternatively or inaddition, the primers may be no more than 50, 40, 30, 28, 26, 25, 24,22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, or fewer nucleotides long.

The primers may have a specific melting temperature. The meltingtemperature of the primer may be at least about 35° C. (°C), 40° C., 41°C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50°C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59°C. 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., or higher. Themelting temperature of the primer may be no more than about 35° C., 40°C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49°C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58°C., 59° C. 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., or lower.

In some cases, primers are ligated together to form a longer primer. Thelonger primer may be subsequently used for any PCR reactions, asdescribed elsewhere herein. Ligating together shorter primers into alonger primer may increase the stringency of binding such that thetargeted barcode is enriched. For example, the shorter primers may beable to distinguish mismatches better and thus bind only to sequenceswith perfect or near perfect complementarity. The longer primer may bemore able to accommodate mismatches in sequences and bind to sequencesthat are not the targeted barcode. In some cases, more than 2 primersare ligated together to form a longer primer. For example, at leastabout 3, 4, 5, 6, 7, 8, 9, 10, or more primers may be ligated togetherto form a longer primer.

In some embodiments, the primer comprises an affinity group and theenriched plurality of capture probes comprises the affinity group. Theaffinity group may comprise biotin. The affinity group may comprisestreptavidin or avidin. The affinity group may comprise dioxigenin. Theaffinity group may comprise a protein or polypeptide. The affinity groupmay comprise an antibody or antibody fragment. The affinity group maycomprise a receptor or receptor ligand. The affinity group may comprisea sugar, a monosaccharide, a disaccharide, or a polysaccharide. Theaffinity group may comprise maltose.

Methods of the present disclosure may be used for various applications,such as, processing a single analyte (e.g., RNA, DNA, or protein) ormultiple analytes (e.g., DNA and RNA, DNA and protein, RNA and protein,or RNA, DNA, and protein) from a single cell. For example, multipleanalytes from the biological particle can be processed. The multipleanalytes may be from the single cell. This may enable, for example,simultaneous proteomic, transcriptomic and genomic analysis of the cell.

Hierarchal Barcode Enrichment

In an aspect, the present disclosure provides methods of spatiallydesigning or designating barcodes, such that the position of thebarcodes are specifically identified. In some embodiments, a particularindividual spot or bead may contain more than one spatial barcode,wherein each spatial barcode corresponds to a particular defined regionon the spotted array. For example, multiple spatial barcode sequencesassociated with one particular spot on an array can provide a strongeraddress or attribution to a given location by providing duplicate orindependent confirmation of the location. In some cases, the multiplespatial barcodes represent increasing specificity of the location of theparticular array point. In a non-limiting example, a particular arraypoint may be coded with two different spatial barcodes, wherein eachspatial barcode identifies a particular defined region within the array,and an array point possessing both spatial barcodes identifies thesub-region where two defined regions overlap, e.g.,, such as theoverlapping portion of a Venn diagram. In another non-limiting example,a particular array point may be coded with three or more differentspatial barcodes, wherein the first spatial barcode identifies a firstregion within the array, the second spatial barcode identifies a secondregion, wherein the second region is a subregion entirely within thefirst region (a sub-spatial region), and the third spatial barcodeidentifies a third region, wherein the third region is a subregionentirely within the first and second subregions (a sub-spatial region),and so on. In some embodiments, each capture probe has a unique spatialbarcode, or unique molecular identifier (UMI). In some embodiments, eachcapture probe has a UMI, as well as one or more target barcodescorresponding to one or more regions or sub-spatial regions.

In some embodiments, an array has multiple regions (also sometimesreferred to as “spots” or “locations”) and subregions. In someembodiments, an array has 10 regions or more. In some embodiments, anarray has 20, 30, 40, 50, 60, 70, 80, 90, 100 regions, or more. In someembodiments, an array has 100, 200, 300, 400, 500, 600, 700, 800, 900,1000 regions or more. In some embodiments, an array has 1000, 2000,3000, 4000, 5000, 6000, 7000, 8000, 9000, 1000 regions or more. In someembodiments, the regions are identical in size. In some embodiments, theregions are not identical in size. The regions may take any shape,including but not limited to square, rectangle, circle, oval, pentagon,hexagon, octagon arc, arches, lens, crescent, and any combinationsthereof. In some embodiments, a region of an array has multiplesubregions. In some embodiments, a region of an array has 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000 subregions or more. In some embodiments, the subregions areidentical in size to other subregions. In some embodiments, thesubregions are not identical in size to other subregions. In someembodiments, the subregions are completely surrounded by a largerregion. In some embodiments, subregions are partially surrounded by alarger region. In some embodiments, an array has multiplesub-subregions. In some embodiments, an array has 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000sub-subregions or more. In some embodiments, the sub-subregions areidentical in size to other sub-subregions. In some embodiments, thesub-subregions are not identical in size to other sub-subregions. Insome embodiments, sub-subregions are completely surrounded by a largerregion. In some embodiments, the larger region is a subregion. In someembodiments, the larger region is a region. In some embodiments,sub-subregions are partially surrounded by a larger subregion. In someembodiments, sub-subregions are partially surrounded by a larger region.In some embodiments, individual spots or beads comprise a region. Insome embodiments, individual spots or beads comprise a subregion. Insome embodiments, individual spots or beads comprise a sub-subregion. Insome embodiments, individual spots or beads comprise a regioncorresponding to single cell resolution of the sample.

In some embodiments, the present disclosure provides methods ofenriching a sample comprising more than one target barcoded nucleicacid. The methods may comprise providing a plurality of capture probescomprising a plurality of different spatial barcode sequences,identifying more than one target barcode sequence from the plurality ofdifferent spatial barcode sequences, and enriching capture probescomprising the more than one target barcode sequence. Enriching may beperformed by purifying capture probes comprising the more than onetarget barcode from a plurality of capture probes. Enriching may beperformed by amplifying (i.e. increasing the numbers of) capture probescomprising the more than one target barcode from a plurality of captureprobes. Enriching may be performed by both purifying and amplifying thecapture probe comprising the more than one target barcode from aplurality of capture probes. Enriching may be performed using targetbarcodes associated with smaller regions. Enriching may be performedusing target barcodes of increasing specificity to a particular spot orlocation. For example, enriching may be performed by amplifying and/orpurifying a first plurality of capture probes comprising a targetbarcode that is associated with a first region. The enriching mayfurther comprise amplifying and/or purifying a second plurality ofcapture probes comprising a target barcode that is associated with asecond region that is partially or completely within the first region.The enriching may further comprise amplifying and/or purifying a secondplurality of capture probes comprising a target barcode that isassociated with a second region that is partially within the firstregion. The enriching may further comprise amplifying and/or purifying asecond plurality of capture probes comprising a target barcode that isassociated with a second region that is completely within the firstregion. The enriching may further comprise amplifying and/or purifying athird plurality of capture probes comprising a target barcode that isassociated with a third region that is partially or completely withinthe first and second regions. The enriching may further compriseamplifying and/or purifying a third plurality of capture probescomprising a target barcode that is associated with a third region thatis partially within the first and second regions. The enriching mayfurther comprise amplifying and/or purifying a third plurality ofcapture probes comprising a target barcode that is associated with athird region that is completely within the first and second regions. Theenriching may further comprise amplifying and/or purifying a fourthplurality of capture probes comprising additional target barcodes thatare associated with four or more regions that are partially orcompletely within the first, second, and third regions. The enrichingmay further comprise amplifying and/or purifying a fifth plurality ofcapture probes comprising additional target barcodes that are associatedwith five or more regions that are partially or completely within thefirst, second, third, and fourth regions. The enriching may furthercomprise amplifying and/or purifying additional pluralities of captureprobes comprising additional target barcodes that are associated with 6,7, 8, 9, 10, or more regions that are partially or completely within thepreviously identified regions and/or sub-spatial regions.

In some embodiments, the method may comprise enriching nucleic acidscomprising more than one target barcode sequence. The enriching maycomprise performing a nucleic acid extension reaction using a captureprobe comprising more than one target barcode sequence and a primercomprising a sequence specific for more than one target barcode sequenceto generate an enriched plurality of capture probes comprising more thanone target barcode sequence. The nucleic acid extension reaction may bea polymerase chain reaction (PCR). The nucleic acid extension reactionmay comprise a polymerase. The nucleic acid extension reaction mayresult in amplification of the nucleic acid.

In some embodiments, the method may further comprise performingadditional rounds of PCR on the enriched plurality of capture probescorresponding to the number of spatial barcode sequences of a givenlocation. The additional rounds of PCR may comprise use of additionalprimers comprising a sequence specific for the additional target barcodesequences (e.g., target barcode sequences associated with a subregion orsub-subregion of interest). In some embodiments, the additional primersmay comprise a sequence specific for the spatial barcode sequence of theenriched plurality of capture probes comprising more than one targetbarcode sequence. In some embodiments, the additional primers maycomprise a sequence substantially specific for an additional targetbarcode sequence of the enriched plurality of capture probes comprisingmore than one target barcode sequence. For example, the additionalprimer may comprises a sequence having at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.9% or more sequence complementarity or sequence identity to a targetbarcode sequence of the enriched plurality of capture probes comprisingthe more than one target barcode sequence. In some embodiments, theadditional primers may comprise a sequence specific for a 5′ region of aspatial barcode sequence of the enriched plurality of capture probescomprising more than one target barcode sequence. In some cases, theadditional primer can comprise a sequence that has no (or low) sequencecomplementarity or identity to one or more spatial barcode sequences ofthe enriched plurality of capture probes comprising more than one targetbarcode sequence.

In some embodiments, each spatial barcode sequence of the plurality ofdifferent spatial barcode sequences can identify a capture probe asderived from a single cell. Alternatively or in addition, each spatialbarcode sequence of the plurality of different spatial barcode sequencesmay identify a capture probe as derived from a single partition.

Identifying a Spatial Region of Interest

In some embodiments, identifying a spatial region of interest includesany technique for observing and/or locating an area within a tissue. Insome embodiments, identifying a spatial region of interest includesimmunostaining techniques, including but not limited to,immunohistochemistry techniques. In some embodiments, identifying aspatial region of interest includes microscopy techniques, including butnot limited to, optical microscopy, fluorescence microscopy, andconfocal microscopy. In some embodiments, identifying a spatial regionof interest includes visual inspection. In some embodiments, identifyinga spatial region of interest includes shallow sequencing a spatialregion. In some embodiments, identifying a spatial region of interestincludes shallow sequencing the entire tissue sample.

In at least one embodiment, the present disclosure provides a method forpreparing a composition comprising: providing a substrate comprising afirst oligonucleotide attached to the substrate through its 3′-end orits 5′-end; and preparing by spatially-directed in situ synthesis on theunattached end of the first segment a plurality of secondoligonucleotide segments of 8-mer to 30-mer each having a differentsequence and located in a different region of the substrate.

In at least one embodiment, the method further comprises: attaching aplurality of third oligonucleotide segments to the unattached ends ofthe plurality of second oligonucleotide segments.

In at least one embodiment of the method of the present disclosure thespatially-directed in situ synthesis is light-directed spatialsynthesis.

In at least one embodiment of the method of the present disclosure thespatially-directed in situ synthesis is mechanically-directed spatialsynthesis.

In at least one embodiment of the method of the present disclosure, thefirst oligonucleotide segment is attached to the substrate through its5′-end and the plurality of second oligonucleotide segments are prepared(e.g., generated) by spatially-directed in situ synthesis on the 3′-endof the first segment.

In at least one embodiment of the method of the present disclosure, thefirst oligonucleotide segment is attached to the substrate through its3′-end and the plurality of second oligonucleotide segments are prepared(e.g., generated) by spatially-directed in situ synthesis on the 5′-endof the first segment.

In at least one embodiment, the present disclosure provides a method forpreparing a composition comprising: providing a substrate comprising afirst oligonucleotide segment attached through its 5′-end to thesubstrate, wherein the first oligonucleotide segment phosphodiester andexocyclic amine moieties are protected; preparing by in situ synthesis aplurality of second oligonucleotide segments of 8-mer to 20-mer attachedthrough their 5′-ends to a 3′-end of a first oligonucleotide segment,wherein each of the plurality of second segments comprises a differentsequence that identifies the region of the substrate where it islocated; and ligating a plurality of third oligonucleotide segmentsthrough their 5′-ends to the 3′-ends of the plurality of secondsegments.

In at least one embodiment of the method of the present disclosure theregions of the substrate have an area of less than about 2500 µm², lessthan about 1600 µm², less than about 400 µm², less than about 100 µm²,less than about 50 µm², less than about 25 µm², less than about 15 µm²,less than about 10 µm², less than about 5 µm², less than about 2 µm²,less than about 1 µm², or smaller.

In at least one embodiment of the method of the present disclosure thenumber of second oligonucleotide segment comprising different sequencesis at least about 100,000, at least about 250,000, at least about500,000, at least about 750,000, at least about 1,000,000, at leastabout 1,500,000, at least about 3,000,000, at least about 5,000,000, atleast about 10,000,000, at least about 100,000,000, at least about500,000,000, or at least about 1,000,000,000. In some embodiments, thenumber of second oligonucleotide segment comprising different sequencesis about 100,000 to about 1,000,000,000, about 750,000 to about1,500,000, about 1,000,000 to about 1,000,000,000, about 1,000,000 toabout 10,000,000, or about 1,500,000 to about 5,000,000.

In at least one embodiment of the method of the present disclosure thesecond oligonucleotide segments have a length selected from 8-mer,9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer,18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, 25-mer, 26-mer,27-mer, 28-mer, 29-mer, or 30-mer.

In at least one embodiment of the method of the present disclosure thesecond oligonucleotide segment in each of the different regions of thesubstrate has a purity of at least 75%, at least 80%, at least 85%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or more.

In at least one embodiment of the method of the present disclosure thefirst oligonucleotide segment further comprises protected phosphodiesterand exocyclic amine moieties.

In at least one embodiment of the method of the present disclosure thesubstrate further comprises a complementary oligonucleotide hybridizedto the first oligonucleotide segment.

In at least one embodiment of the method of the present disclosure thefirst oligonucleotide segment has a length of at least about 2-mer, atleast about 5-mer, at least about 8-mer, at least about 10-mer, or atleast about 15-mer at least about 18-mer, at least about 20-mer, atleast about 22-mer, or at least about 25-mer, at least about 27-mer, atleast about 30-mer, at least about 33-mer, or at least about 35-mer. Insome embodiments the first oligonucleotide segment has a length of about2-mer to about 45-mer, about 5-mer to about 40-mer, about 15-mer toabout 40-mer about 25-mer to about 40-mer, about 25-mer to about 35-mer,or about 27-mer to about 33-mer.

In at least one embodiment of the method of the present disclosure themethod further comprises preparing the first oligonucleotide segment offthe substrate and subsequently attaching it to the substrate. In someembodiments the phosphodiester and exocyclic amine moieties of the firstoligonucleotide segment are protected prior to attaching it to thesubstrate. In some embodiments the phosphodiester and exocyclic aminemoieties of the first oligonucleotide segment are protected afterattaching it to the substrate.

In at least one embodiment of the method of the present disclosure themethod further comprises preparing the first oligonucleotide segment bysynthesis on the substrate. In some embodiments the synthesis of thefirst oligonucleotide segment on the substrate is by light-directedspatial synthesis. In some embodiments the synthesis of the firstoligonucleotide segment on the substrate is not spatially-directed.

In at least one embodiment of the method of the present disclosure thefirst oligonucleotide segment sequence comprises a cleavage site. Insome embodiments, the cleavage site comprises a poly-U sequence.

In at least one embodiment of the method of the present disclosure thefirst oligonucleotide segment is attached to the substrate through alinker. In some embodiments, the linker comprises a chemical groupselected from: silane, amine, peptide, polyethylene glycol, biotin,desthiobiotin, streptavidin, thiol, acrydite, azide, digoxigenin,hexynyl, or a combination thereof. In some embodiments, the linker is abranched linker or a dendrimeric linker.

In at least one embodiment of the method of the present disclosure thatfurther comprises attaching a plurality of third oligonucleotidesegments, the first oligonucleotide segment is attached to the substratethrough its 5′-end, the plurality of second oligonucleotide segments areprepared by spatially-directed in situ synthesis on the 3′-end of thefirst segment, and the plurality of third oligonucleotide segments areattached through their 5′-end to the 3′-end of the plurality of secondsegments.

In at least one embodiment of the method of the present disclosure thatfurther comprises attaching a plurality of third oligonucleotidesegments, the plurality of third oligonucleotide segments are attachedby ligation. In some embodiments, protected moieties of the first andsecond oligonucleotide segments are deprotected prior to ligating theplurality of third oligonucleotide segments.

In at least one embodiment of the method of the present disclosure thatfurther comprises attaching a plurality of third oligonucleotidesegments, the plurality of third oligonucleotide segments have a lengthof at least about 5-mer, at least about 7-mer, at least about 9-mer, atleast about 13-mer, at least about 20-mer, at least about 25-mer, atleast about 27-mer, at least about 30-mer, at least about 33-mer, or atleast about 35-mer. In some embodiments, the plurality of thirdoligonucleotide segments have a length of about 5-mer to about 40-mer,5-mer to about 20-mer, 5-mer to about 10-mer, about 10-mer to about35-mer, about 15-mer to about 30-mer, about 25-mer to about 35-mer, orabout 27-mer to about 33-mer.

In at least one embodiment of the method of the present disclosure thatfurther comprises attaching a plurality of third oligonucleotidesegments, each of the plurality of third oligonucleotide segmentscomprise a different 5-mer to 20-mer random sequence at the 5′-end.

In at least one embodiment of the method of the present disclosure thatfurther comprises attaching a plurality of third oligonucleotidesegments, each of the plurality of third oligonucleotide segmentscomprises an identical capture sequence of at least 6-mer at the 3′-end.In some embodiments, the capture sequence comprises a poly-T or a poly-Usequence.

In at least one embodiment the present disclosure also provides acomposition comprising a plurality of oligonucleotides attached to aplurality of regions on a substrate, wherein each oligonucleotidecomprises: a first oligonucleotide segment of 2-mer to 50-mer attachedto the substrate through its 5′-end; and a second oligonucleotidesegment of 8-mer to 30-mer that is attached to the 3′-end of the firstsegment through its 5′-end, wherein the second segment comprises asequence that identifies the region where it is located.

In at least one embodiment of the composition each oligonucleotidefurther comprises: a third oligonucleotide segment of 11-mer to 50-merthat is attached to the 3′-end of the second segment through its 5′-end,wherein the third segment comprises a 5-mer to 20-mer random sequence atits 5′-end and a 6-mer to 38-mer capture sequence of at its 3′-end.

In at least one embodiment of the composition of the present disclosurethe regions of the substrate have an area of less than about 2500 µm²,less than about 1600 µm², less than about 400 µm², less than about 100µm², less than about 50 µm², less than about 25 µm², less than about 15µm², less than about 10 µm², less than about 5 µm², less than about 2µm², less than about 1 µm², or smaller.

In at least one embodiment of the composition of the present disclosurethe number of second oligonucleotide segment comprising differentsequences is at least about 100,000, at least about 250,000, at leastabout 500,000, at least about 750,000, at least about 1,000,000, atleast about 1,500,000, at least about 3,000,000, at least about5,000,000, at least about 10,000,000, at least about 100,000,000, atleast about 500,000,000, or at least about 1,000,000,000. In someembodiments, the number of second oligonucleotide segment comprisingdifferent sequences is about 100,000 to about 1,000,000,000, about750,000 to about 1,500,000, about 1,000,000 to about 1,000,000,000,about 1,000,000 to about 10,000,000, or about 1,500,000 to about5,000,000.

In at least one embodiment of the composition of the present disclosurethe second oligonucleotide segments have a length selected from 8-mer,9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer,18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, 25-mer, 26-mer,27-mer, 28-mer, 29-mer, or 30-mer.

In at least one embodiment of the composition of the present disclosurethe second oligonucleotide segment in each of the different regions ofthe substrate has a purity of at least 95%, at least 96%, at least 97%,at least 98%, at least 99%, or more.

In at least one embodiment of the composition of the present disclosurethe first and second oligonucleotide segments further comprise protectedphosphodiester and exocyclic amine moieties.

In at least one embodiment of the composition of the present disclosurethe length of the oligonucleotide in each region is at least 10-mer, atleast 20-mer, at least 30-mer, at least 40-mer, at least 50-mer, or atleast 60-mer. In some embodiments the length of the oligonucleotide ineach region is from 40-mer to 100-mer, from 50-mer to 90-mer, from60-mer to 80-mer or 10-mer to 40-mer.

Generating Spatial Arrays

In at least one embodiment of the compositions and methods of thepresent disclosure, the plurality of oligonucleotides is attached to asubstrate by any suitable means that immobilizes the oligonucleotide byeither its 3′ or 5′ end in a region of the substrate and allows theunattached end to be available for further synthesis. Further synthesiscan include in situ base-by-base step-wise synthesis (e.g.,phosphoramidite chemistry) or biosynthesis via enzyme reactions (e.g.,DNA polymerization or reverse transcription). Additionally, the means ofattachment should also be suitable to allow at least a portion of theoligonucleotide to be capable of hybridization.

In at least one embodiment of the compositions and methods of thepresent disclosure, the plurality of oligonucleotides is attached to asubstrate by a covalent linkage. Accordingly, in at least oneembodiment, the plurality of oligonucleotides are attached to thesubstrate by a chemical reaction that forms one or more covalent bondsbetween a functional group on the substrate (or a linker group attachedto the substrate) and a functional group on the 3′- or 5′-end of theoligonucleotide (or a linker group attached to the 3′ or 5′-end of theoligonucleotide). Such functional groups can include a naturallyoccurring nucleotide functional group, e.g. a terminal hydroxyl group,or a non-natural functional group introduced during oligonucleotidesynthesis, such as a 5′-amine group. Reagents and techniques forchemically modifying substrate materials and/or nucleotides withfunctional groups that are useful in the methods of the presentdisclosure are well known in the art. For example, functional groupsthat may be introduced on a substrate material that allow for attachmentof an oligonucleotide (e.g., 5′-amine modified oligonucleotide) to asubstrate include linkers with available carboxy, aldehyde or epoxygroups. Other functionalized linker compounds are known to the personskilled in the art.

The oligonucleotides of the plurality of oligonucleotides to be attachedto a substrate in preparing a spatial array of the present disclosurecan be understood as having at least three segments: a firstoligonucleotide segment of 2-mer to 40-mer that is attached to thesubstrate through either its 3′-end or its 5′-end; a secondoligonucleotide segment that is attached to the unattached end of thefirst segment, and comprises a barcode sequence of about 8-mer to 30-merin length that indicates the region on the substrate where the secondsegment is located; and a plurality of third oligonucleotide segments of5-mer to 50-mer attached to the second segment, wherein the thirdsegment comprises a capture sequence at its 3′-end.

FIGS. 31A-C depict at least one embodiment of a method for preparing anexemplary spatial array composition of the present disclosure. First, asshown in FIG. 31A, a substrate is provided that comprises a firstoligonucleotide segment attached through its 5′-end to the substrate. Inat least one embodiment, the first oligonucleotide segment comprises asequence that is identical across all of the plurality of the differentoligonucleotides attached to the substrate. In some embodiments, thefirst oligonucleotide segment can be understood as a commonoligonucleotide “lawn” across the surface of the substrate that does notdiffer in sequence in the various regions of the substrate. The free,unattached end of the first oligonucleotide segment, thus, effectivelyacts as the starting point on the spatial array for thespatially-directed in situ synthesis of the second oligonucleotidesegment that comprises the barcode sequence.

In at least one embodiment, the length of the first oligonucleotidesegment is the same across the surface of the substrate. While typicallynot critical to the function of the spatial array, in at least oneembodiment, the length of the first oligonucleotide sequence should beat least about 2-mer. Furthermore, in some embodiments the length of thefirst oligonucleotide segment is at least 5-mer, at least about 10-mer,at least about 15-mer, at least about 20-mer, at least about 25-mer, atleast about 27-mer, at least about 30-mer, at least about 33-mer, or atleast about 35-mer. Similarly, in some embodiments the firstoligonucleotide segment has a length of about 2-mer to about 40-mer,about 5-mer to about 40-mer, about 10-mer to about 30-mer, about 25-merto about 35-mer, about 20-mer to about 40-mer, about 25-mer to about40-mer, about 25-mer to about 35-mer, or about 27-mer to about 33-mer.

As further depicted in FIG. 31A, in at least one embodiment, the firstoligonucleotide segment comprises phosphodiester and exocyclic aminemoieties that are protected. Protection of the phosphodiester andexocyclic amine moieties of the first oligonucleotide segment allows foroptimal in situ synthesis of the second oligonucleotide segments thatcomprise the barcode sequence. Suitable protecting groups forphosphodiester and exocyclic amine moieties are well-known in the artand include the protecting groups employed in standard oligonucleotidesynthesis using phosphoramidite chemistry.

The present disclosure describes a variety of embodiments for preparinga substrate having attached thereto a first oligonucleotide segment,wherein the first oligonucleotide segment comprises protectedphosphodiester and exocyclic amine moieties (see e.g., embodiments ofFIG. 32A-C to 36A-C).

In at least one exemplary embodiment, depicted in FIGS. 32A-C, themethod further comprises preparing the first oligonucleotide segment exsitu (e.g., on a separate solid support using solid phase automatedsynthesis), cleaving it from the separate solid support with theprotecting groups intact (see FIG. 32A), and subsequently attaching theex situ synthesize oligonucleotide with protecting groups it to thesubstrate (see FIG. 32B). A free hydroxyl at the unattached end isavailable for subsequent spatially-directed in situ synthesis of asecond oligonucleotide segment comprising a barcode sequence (see FIG.32C).

In at least another exemplary embodiment, depicted in FIGS. 33A-B, themethod further comprises preparing the first oligonucleotide segment insitu directly the solid support using base-by-base phosphoramiditesynthesis chemistry with phosphodiester and exocyclic amine protectinggroups. Such a method can be use standard solid-phase oligonucleotidesynthesis chemistry to provide a substrate with first oligonucleotidesegments attached thereto by the 3′-end and having protecting groups. Inat least one embodiment, as depicted in FIG. 33A, oligonucleotidesynthesis chemistry in a 5′-to-3′ direction can be carried out toprovide the protected first oligonucleotide segments attached to thesubstrate through the 5′-end. In both cases, the free hydroxyl at theunattached end is available for subsequent spatially-directed in situsynthesis of a second oligonucleotide segment comprising a barcodesequence (see FIG. 33C).

Second, as depicted in FIG. 31B, a plurality of second oligonucleotidesegments of 8-mer to 30-mer are prepared by spatially-directed in situsynthesis extending from the free end of the first oligonucleotidesegment. As shown in FIG. 31B, the spatially-directed in situ synthesisis a light-directed spatial synthesis which is carried out in the5′-to-3′ direction. This in situ synthesis method results in a pluralityof second oligonucleotide segments each attached through its 5′-end to a3′-end of a first oligonucleotide segment. FIG. 31B is a schematicrepresentation of only two distinct regions each having four individualoligonucleotide molecules. Each region has a different secondoligonucleotide segment sequence, and all of the second oligonucleotidesegment molecules in a region ideally should have the same sequence,although as discussed further below, less than 100% yield in the in situsynthesis process results in less than perfect purity (or homogeneity)of oligonucleotide sequence in a region.

In at least one embodiment of the present disclosure, spatially-directedsynthesis of a plurality of oligonucleotides on a substrate in spatiallydistinct regions is carried out using light-directed synthesis or bymechanically-directed synthesis methods. A spatially-directed in situsynthesis method inherently correlates each sequence synthesized withthe location of synthesis. In the schematic illustration of FIG. 31B, alight-directed spatial synthesis is used to synthesize a secondoligonucleotide segment in a base-by-base step-wise fashion in eachregion. The region where each nucleotide base is added is controlled bythe precise location (and duration) of spatially-directed illuminationof the substrate. The direction and duration of illumination can beprecisely controlled, for example with maskless array synthesis (MAS)technology using computer-controlled digital micromirror devices (DMD),to provide distinct regions of 1 µm², or even smaller. It is only atthese precisely illuminated regions on the substrate where deprotectionof a photo-labile protecting group occurs, thereby allowing coupling ofa nucleoside phosphoramidite that provides the next base in theoligonucleotide sequence.

Light-directed spatial in situ synthesis provides optimal control in thesynthesis of high-fidelity second oligonucleotide segment barcodesequences at precise region locations on a substrate. Light-directedspatial synthesis of oligonucleotides refers to synthetic methods usingphotolabile nucleoside protecting groups and spatially-directedillumination to effect selective deprotection and base-by-base synthesisat specific locations on a substrate. Light-directed spatial synthesisof oligonucleotides uses an iterative cycle of phosphoramidite-basedcoupling chemistry with photo-labile protecting groups rather thandimethoxytrityl (DMT) protecting groups. The synthesis cycle, thus, isvery similar to that used in standard solid-phase phosphoramiditesynthesis of oligonucleotides with the following general steps: (1)photo-deprotection; (2) activation and coupling; (3) capping; and (4)drying. The drying step (4) can involve blowing helium to dry thesubstrate surface after coupling and acetonitrile wash but before thenext photodeprotection step. Techniques, protocols, reagents, materials,and other instrumentation useful for light-directed spatial synthesis ofhigh-density oligonucleotide microarrays are well-known in the art andcommercially available. For example, US Pat. Nos. 7,844,940, 8,030,477,PCT Publ. No. WO 99/63385A1, US Pat. Publ. No. 20150246336A1, Agbavwe etal. “Efficiency, error and yield in light-directed maskless synthesis ofDNA microarrays.” Journal of nanobiotechnology 9.1 (2011): 57; and Sacket al. “Express photolithographic DNA microarray synthesis withoptimized chemistry and high-efficiency photolabile groups.” Journal ofnanobiotechnology 14.1 (2016): 14, describe materials and methods thatcan be used in the light-directed spatial synthesis methods of thepresent disclosure.

The photo-deprotection step involves spatially-directed illumination ofthe substrate and is the step that ultimately defines the size andlocation of a region. Efficiency and accuracy of the photo-deprotectionstep in defining the regions on the substrate where specificoligonucleotides are synthesized depends largely on the illuminationtechniques and the photo-labile groups used. Photo-labile groups andillumination techniques useful for light-directed spatial synthesismethods are well-known and commercially available (e.g., fromSigma-Aldrich, St. Louis, MO, USA). For example, photo-labile protectinggroups that can be used to prepare spatial array compositions of thepresent disclosure are described in US Pat. Publ. No. 20150246336A1;Agbavwe et al. “Efficiency, error and yield in light-directed masklesssynthesis of DNA microarrays.” Journal of nanobiotechnology 9.1 (2011):57; Kretschy et al. “Next-Generation o-Nitrobenzyl Photolabile Groupsfor Light-Directed Chemistry and Microarray Synthesis.” AngewandteChemie International Edition 54.29 (2015): 8555-8559; Sack et al.“Express photolithographic DNA microarray synthesis with optimizedchemistry and high-efficiency photolabile groups.” Journal ofnanobiotechnology 14.1 (2016): 14; Lietard et al., “High-Density RNAMicroarrays Synthesized In Situ by Photolithography.” Angew. Chem. Int.Ed. 57 (2016): 15257-15261; and Hölz et al., “High-Efficiency Reverse(5′→ 3′) Synthesis of Complex DNA Microarrays.” Scientific reports 8.1(2018): 15099.

In at least one embodiment, the photo-labile protecting groups useful inthe light-direct spatial synthesis methods of the present disclosureinclude NPPOC, Bz-NPPOC, and SPh-NPPOC which are shown in Table 1.

TABLE 1 Exemplary Photo-labile Protecting Groups Name Structure (e.g.,protecting 3′-OH of nucleoside) NPPOC

Bz-NPPOC

SPh-NPPOC

The schematic illustration of FIG. 31B depicts light-directed spatialsynthesis of second oligonucleotide segments in the 5′-to-3′ direction.The resulting plurality of the second oligonucleotide segments are thusattached through their 5′-ends to the free 3′-ends of the firstoligonucleotide segment on the substrate. Accordingly, in at least oneembodiment the light-directed spatial synthesis of the secondoligonucleotides is carried out in the 5′-to-3′ direction. Techniques,reagents, and photo-labile protecting groups useful for synthesis in the5′-to-3′ direction include those described in e.g., Hölz et al.,“High-Efficiency Reverse (5′→ 3′) Synthesis of Complex DNA Microarrays.”Scientific reports 8.1 (2018): 15099. In at least one embodiment,light-directed spatial synthesis of a plurality of secondoligonucleotide segments can be carried out using one or more of thephoto-labile protecting groups of Table 1 to protect the 3′—OH. In atleast one embodiment, the photo-labile protecting group used inlight-directed spatial synthesis of the second oligonucleotide segmentis Bz-NPPOC.

In at least one embodiment, the spatially-directed in situ synthesis canbe carried out using mechanically-directed spatial synthesis.Mechanically-directed spatial synthesis of oligonucleotides refers tomethods that use computer-controlled liquid dispensing and/or placementtechnology to mechanically direct nucleoside phosphoramidite reagents atdistinct locations on a substrate, and thereby synthesizing anoligonucleotide using standard solid-phase phosphoramidite chemistry ata distinct region on a substrate. A range of technologies for mechanicalspatially-directed in situ synthesis of oligonucleotides are well-knownin the art.

The schematic illustration of FIG. 31B depicts only two regions, howeverit is contemplated that a spatial array composition made by the methodsof the present disclosure can include millions of different regions on asubstrate, each comprising a different second oligonucleotide segmentbarcode sequence. In at least one embodiment, the number of secondoligonucleotide segments comprising different sequences is at leastabout 100,000, at least about 250,000, at least about 500,000, at leastabout 750,000, at least about 1,000,000, at least about 1,500,000, atleast about 3,000,000, at least about 5,000,000, at least about10,000,000, at least about 100,000,000, at least about 500,000,000, orat least about 1,000,000,000. In some embodiments, the number of secondoligonucleotide segment comprising different sequences is about 100,000to about 1,000,000,000, about 750,000 to about 1,500,000, about1,000,000 to about 1,000,000,000, about 1,000,000 to about 10,000,000,or about 1,500,000 to about 5,000,000.

In order for a spatial array to accommodate these large numbers ofdifferent oligonucleotides on a substrate, it also is contemplated thatthe areas of the regions need to be small. Accordingly, in at least oneembodiment of the method of the present disclosure the regions of thesubstrate have an area of less than about 2500 µm², less than about 1600µm², less than about 400 µm², less than about 100 µm², less than about50 µm², less than about 25 µm², less than about 15 µm², less than about10 µm², less than about 5 µm², less than about 2 µm², less than about 1µm², or smaller.

In at least one embodiment, the methods of the present disclosurecontemplate the spatially-directed in situ synthesis of secondoligonucleotide segments comprising millions of different barcodesequences each in a different region. In order to accommodate millionsof distinct regions each with a distinct barcode sequence, the presentdisclosure contemplates that the second oligonucleotide segments have alength range of 8-mer to 30-mer. A length range of 8-mer to 30-merallows for the plurality of oligonucleotides to have extremely largenumber of distinct oligonucleotides that are indexed for their locationon the substrate. For example, in the case of an 8-mer sequence, thereare 48 or 65,536 different possible barcode sequences that can be usedto identify distinct locations on a substrate. In the case of a 12-mersequence, there are 412 or 16,777,216 different possible barcodesequences that can be used to identify distinct locations on asubstrate. Accordingly, in at least one embodiment of the method of thepresent disclosure the second oligonucleotide segments have a lengthselected from 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer,15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer,24-mer, 25-mer, 26-mer, 27-mer, 28-mer, 29-mer, or 30-mer.

In order to provide for optimal functionality of a spatial array, thecorrelation of the barcode sequences with the locations should be high.Accordingly, the spatially-directed in situ synthesis of the barcodesequence should result in high fidelity or purity of barcode sequencesin each of the regions. It is an advantage of the spatially-directedsynthesis methods of the present disclosure that the secondoligonucleotide segments are prepared on a substrate with high fidelity.Generally, the fidelity (or sequence purity) of oligonucleotidesequences prepared by step-wise solid-phase synthesis decreases as thelength of the oligonucleotide being synthesized increases. For example,assuming a spatially-directed in situ synthesis having a couplingefficiency of 99.5%, the fidelity of 12-mer barcode sequences in aregion is ~95%. In some embodiments (e.g., as in FIGS. 32A-C), the useof spatially-directed in situ synthesis to prepare only the 8-mer to30-mer second oligonucleotide segments results in barcode sequences ofhigh-fidelity. Accordingly, in at least one embodiment of the methods ofthe present disclosure the resulting second oligonucleotide segmentsynthesized in each of the different regions of the substrate has apurity of at least 75%, at least 80%, at least 85%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or more.

In at least one embodiment, the second oligonucleotide segment is uniquefor each region of the substrate. Additionally, in some embodiments, thesecond oligonucleotide segment barcode sequence comprises a sequencethat uniquely identifies it from the plurality of other barcodesequences on the spatial array but does not interfere with the capturesequences of the oligonucleotides that target binding to nucleic acidsin a sample. Unique barcode sequences may be generated using randomsequence generation, along with stringent filtering of sequences bymapping to the genomes of all common reference species using desired Tmintervals, and GC content, to minimize interference with capturesequence binding to their targets, and to optimize the difference fromthe other barcode sequences. Thus, in at least one embodiment, thebarcode sequences of the spatial array are selected such that desiredtarget nucleic acids of the sample applied to the array do not hybridizespecifically to the barcode sequence of the second oligonucleotidesegment. For example, in some embodiments, the barcode sequences of thespatial array have less than 80% sequence identity to the targetednucleic acid sequences in the sample applied to the spatial array. Insome embodiments, barcode sequence has less than 70% sequence identity,less than 60% sequence identity, less than 50% sequence identity, orless than 40% sequence identity to the nucleic acids in the sampletargeted for capture by the plurality oligonucleotide sequences.

Third, as depicted in FIG. 31C, a plurality of third oligonucleotidesegments are attached to the unattached end of the second segment. In atleast one embodiment, the third oligonucleotide segment is in the sameorientation as the second segment. Thus, if the second segment isattached to the first segment through its 5′-end (in a 5′-to-3′orientation relative to the substrate) then the third oligonucleotidesegment is attached through its 5′-end to the 3′-end of the secondsegment. As exemplified in FIG. 31C, in at least one embodiment, thethird oligonucleotide segments are attached through their 5′-ends to the3′-ends of the plurality of second segments. In some embodiments, thethird oligonucleotide segment has a length of about 5-mer to about40-mer and comprises a capture sequence capable of hybridizing to anucleic acid in a sample. As described further herein, in someembodiments, the third oligonucleotide segment comprises a free 3′—OHand is capable of hybridizing to a complementary nucleic acid in asample and then undergoing biosynthetic (e.g., polymerase catalyzed)extension from it 3′—OH.

In at least one embodiment, the plurality of third oligonucleotidesegments have a length of at least about 5-mer, at least about 10-mer,at least about 15-mer, at least about 20-mer, at least about 27-mer, atleast about 30-mer, at least about 33-mer, or at least about 35-mer. Insome embodiments, the plurality of third oligonucleotide segments have alength of about 5-mer to about 40-mer, about 10-mer to about 40-mer,about 20-mer to about 40-mer, about 25-mer to about 35-mer, or about27-mer to about 33-mer.

The 5-mer to 40-mer length of a third oligonucleotide segment makes itsattachment by in situ synthesis on the ends of the secondoligonucleotide segments problematic. The accumulating errors during insitu synthesis would compromise the ability of any capture sequences tofunction effectively on the spatial array. Accordingly, in at least oneembodiment of the methods of the present disclosure, the thirdoligonucleotide segments are prepared off the substrate and attached asa whole segment by chemical coupling or ligation to the plurality ofsecond oligonucleotide segments. In at least one embodiment, theplurality of third oligonucleotide segments are attached by ligation. Insome embodiments, protected moieties of the first and secondoligonucleotide segments are deprotected prior to ligating the pluralityof third oligonucleotide segments.

In at least one embodiment where the method of the present disclosurefurther comprises attaching a plurality of third oligonucleotidesegments, the first oligonucleotide segment is attached to the substratethrough its 5′-end, the plurality of second oligonucleotide segments areprepared by spatially-directed in situ synthesis on the 3′-end of thefirst segment, and the plurality of third oligonucleotide segments areattached through their 5′-end to the 3′-end of the plurality of secondsegments.

Alternatively, in at least one embodiment where the method of thepresent disclosure further comprises attaching a plurality of thirdoligonucleotide segments, the first oligonucleotide segment is attachedto the substrate through its 3′-end, the plurality of secondoligonucleotide segments are prepared by spatially-directed in situsynthesis on the 5′-end of the first segment, and the plurality of thirdoligonucleotide segments are attached through their 3′-end to the 5′-endof the plurality of second segments.

In at least one embodiment, the third oligonucleotide segment comprisesa capture sequence capable of hybridizing to a nucleic acid sequence ina sample. In at least one embodiment, the capture sequence is located atthe free 3′-end of the third oligonucleotide segment which is orientedwith its 5′-end attached to the second oligonucleotide segment. In thisorientation the free 3′-end can be extended (e.g. by template dependentpolymerization). It is contemplated that in at least one embodiment, thecapture sequence of the third segment comprises a sequence capable ofhybridizing to a specific type of target nucleic acid that is present ina sample. For example, the capture sequence of the third segment can bedesigned to selectively capture mRNA by hybridization to the poly-Atail. Accordingly, in at least one embodiment, the capture sequence ofthe third oligonucleotide segment comprises a poly-T oligonucleotide.Alternatively, in some embodiments, the capture sequence may comprisenucleotides which are functionally analogous to poly-T and capable ofbinding selectively to poly-A, such as a poly-U oligonucleotide. In atleast one embodiment, the capture sequence of the third oligonucleotidecomprises is a poly-T or poly-U sequence of at least 5-mer to 40-mer inlength. In some embodiments, the capture sequence comprises a poly-T orpoly-U sequence of at least 5-mer, 10-mer, at least 15-mer, at least20-mer, at least 25-mer, at least 30-mer, at least 35-mer, or at least40-mer in length.

In some embodiments, it is contemplated that the capture sequence can bedesigned to selectively bind to a desired sub-type or subset of nucleicacid targets suspected of being present in a sample. For example, aparticular type of RNA, such as mRNA, microRNA or rRNA, or a particularmRNA species that corresponds to a particular group of genes. Thus,capture sequence of the third oligonucleotide segment may include asequence that specifically binds to a particular target gene or set ofgenes, and can be based on a particular sequence motif sequence orconserved sequence according to methods well known in the art.

In some embodiments, the third segments of the plurality of theoligonucleotide can also comprise a random sequence. As is well-known inthe art, such random sequences (e.g. random hexamers or nonamers) areeffective for the capture nucleic acid targets by selective binding.Accordingly, it is contemplated that the capture sequence of the thirdsegments of the plurality of oligonucleotides can comprise a randomsequence, where the random sequences are at least 6-mers, at least8-mers, at least 10-mers, at least 12-mers, at least 14-mers, at least16-mers, or longer. In some embodiments, the plurality of thirdoligonucleotide segments can include random sequences together with apoly-T or poly-U capture sequence. Accordingly, where a thirdoligonucleotide comprises a poly-T or poly-U capture sequence, it mayalso comprise a random sequence. In some embodiments, where the thirdsegment is in a 5′-to-3′ orientation, the random sequences are locatedat the 5′-end of the third segment adj acent to the 3-end of the secondsegment comprising the barcode sequence.

In at least one embodiment, the first, second, and third oligonucleotidesegments are linked via phosphodiester bonds. Additionally, in someembodiments, it is contemplated that the oligonucleotide segments can beattached via non-natural oligonucleotide linkages, such asmethylphosphonate or phosphorothioate bonds, or via other non-naturalbiocompatible linkages such as click-chemistry linkages. The bondslinking the segments can be formed by synthetic phosphoramidite couplingchemistry, by a synthetic click-chemistry, by a ligation reaction, byenzymatic biosynthesis of nucleic acid polymers (e.g., using apolymerase or transcriptase), or a combination of bond formingreactions. For example, in at least one embodiment, the secondoligonucleotide segment is attached to the first oligonucleotide segmentby standard phosphoramidite chemistry forming a phosphodiester linkage,and the third oligonucleotide segment is attached to the unattached endof the second segment via a ligation reaction.

The plurality of oligonucleotides on a spatial array can be attached(e.g., covalently bound or immobilized) to the substrate via either the3′-end or 5′-end of the oligonucleotide. The attachment of anoligonucleotide to a substrate of a spatial array through a covalentbound can be either direct (e.g., substrate to 3′-end ofoligonucleotide) or indirect (e.g., substrate to linker molecule to3′-end of oligonucleotide). Typically, for commercially available DNAmicroarrays, the oligonucleotides are attached via a 3′ linkage, therebyleaving a free 5′-end. The free 5′-end can be used for further synthesison the oligonucleotide strand. However, oligonucleotides attached to thesubstrate via the 5′-end, provide an advantage in having their 3′-endsavailable for further biosynthetic extension (e.g., using polymerase ortranscriptase) as described elsewhere herein.

In at least one embodiment, the first oligonucleotide segment isattached to the substrate through its 5′-end, the second oligonucleotidesegment is attached to the 3′-end of the first segment through its5′-end, and the third segment is attached to the 3′-end of the secondsegment through its 5′-end.

In at least one embodiment, the first oligonucleotide segment isattached to the substrate through its 3′-end; the second oligonucleotidesegment that is attached to the 5′-end of the first segment through its3′-end, and the third segment is attached to the 5′-end of the secondsegment through its 3′-end.

In at least one embodiment of the composition of the present disclosurethe length of the oligonucleotide in each region is at least 10-mer, atleast 20-mer, at least 30-mer, at least 40-mer, at least 50-mer, or atleast 60-mer. In some embodiments the length of the oligonucleotide ineach region is from 5-mer to 100-mer, from 10-mer to 100-mer, from20-mer to 100-mer, from 40-mer to 100-mer, from 50-mer to 90-mer, orfrom 60-mer to 80-mer.

As described elsewhere herein, some embodiments of the spatial arrays ofthe present disclosure, the first oligonucleotide segment of the spatialarray comprises a 2-mer to 40-mer having the common sequence attachedacross all, or a substantial contiguous portion, of the surface of thesubstrate. Thus, the step of attaching the common first oligonucleotidesegment to the substrate does not need to be spatially directed.Accordingly, a variety of different embodiments can be used to attachprepare a substrate having a common first oligonucleotide segment of2-mer to 40-mer attached thereto.

As illustrated by FIGS. 32A-C, in at least one embodiment, a firstoligonucleotide segment is prepared ex situ by standard automatedsolid-phase oligonucleotide synthesis in a 3′-to-5′ direction usingphosphoramidite chemistry. However, as described elsewhere herein,protection of the phosphodiester and exocyclic amine moieties of thefirst oligonucleotide segment facilitates the spatially-directed in situsynthesis of the second oligonucleotide segment on the unattached end ofthe first oligonucleotide segment. Thus, as shown in FIG. 32A, in atleast one embodiment, the first oligonucleotide segment is synthesizedex situ using standard techniques, however, the synthesizedoligonucleotide is cleaved from the solid-support without deprotectingthe phosphodiester and exocyclic amine moieties that are protectedduring synthesis. The protected common oligonucleotide used for thefirst segment is attached to the substrate with the protecting groupsintact as show in FIG. 32B.

The standard oligonucleotide synthesis method using phosphoramiditechemistry was developed in the 1980s and later enhanced with solid-phasesupports and automation. It the method used by commercial synthesis ofDNA and RNA oligonucleotides. Generally, solid-phase oligonucleotidesynthesis proceeds in the 3′→ 5′ direction rather than 5′→ 3′ directionof natural biosynthesis. The solid-phase phosphoramidite synthesis cyclegenerally comprises four steps: (1) detritylation; (2) coupling; (3)oxidation; and (4) capping. Briefly, the four steps in the synthesiscycle are as follows. In step (1), “detritylation,” a 5′-DMT protectinggroup is removed from the first, solid-support-linked nucleoside. Instep (2), “coupling,” the de-protected 5′—OH of the first,solid-support-linked nucleoside attacks the phosphorus of the incomingsecond nucleoside to form a phosphite triester. In step (3),“oxidation,” the relatively unstable phosphite triester is converted toa stable phosphate triester. In step (4), prior to moving to step (1) ofthe next cycle, “capping,” is carried out wherein unreacted 5′—OH groupsare acetylated, thereby preventing elongation of sequences with deletionmutations.

In at least one embodiment of the ex situ synthesis method, the finalmonomer unit added to the 5′-end during ex situ synthesis of the firstoligonucleotide segment can comprise a non-natural nucleoside thatfacilitates attachment or immobilization of the first oligonucleotidesegment to the substrate or linkers attached to the substrate via its5′-end. Alternatively, in some embodiments of the ex situ synthesismethod, the first monomer unit added to the 3′-end during ex situsynthesis of the first oligonucleotide segment can comprise anon-natural nucleoside that upon cleavage from the synthesis solidsupport can facilitate attachment or immobilization of the firstoligonucleotide segment to the substrate or linkers attached to thesubstrate via the 3′-end.

In at least one embodiment, the first oligonucleotide segment attachedto the substrate is prepared by in situ synthesis on the substrate.Generally, because the sequence of the first oligonucleotide segment isthe same across all regions of the substrate, it is not necessary to usespatially-directed in situ synthesis in such an embodiment. For example,in situ synthesis of the first oligonucleotide can be carried out on thesubstrate using standard solid-phase phosphoramidite chemistry andreagents. As noted above, maintaining protection of the phosphodiesterand exocyclic amine moieties of the first oligonucleotide segmentfacilitates the spatially-directed in situ synthesis of the secondoligonucleotide segment on the unattached end of the firstoligonucleotide segment. Accordingly, in at least one embodiment, it iscontemplated that the in situ synthesis first oligonucleotide segment onthe substrate is carried out without the final step of deprotecting thephosphodiester and exocyclic amine moieties before the second segment issynthesized.

FIGS. 33A-B illustrate at least one embodiment of a method for in situsynthesis of a common first oligonucleotide segment directly on thesubstrate. The synthesis is carried out using step-wise solid-phasephosphoramidite chemistry but without final cleavage or deprotectionsteps. As shown in FIG. 33A, in this embodiment, the synthesis wouldresult in a first oligonucleotide segment attached across the surface ofthe substrate with protected phosphodiester and exocyclic aminemoieties. Following this in situ synthesis of the first oligonucleotidesegment with protective groups intact, light-directed spatial synthesisof the second oligonucleotide segment to the 3′-ends of the firstsegments can be carried out as shown in FIG. 33B.

In at least one embodiment, the first oligonucleotide segment attachedto the substrate is prepared by spatially-directed in situ synthesis onthe substrate. The spatially-directed oligonucleotide synthesis methodused can include light-directed spatial synthesis andmechanically-directed spatial synthesis described elsewhere herein. Inthis embodiment, the spatially-directed synthesis can be used inessentially the same way as the in situ standard oligonucleotidesynthesis for the preparation of the first oligonucleotide segmenthaving a single sequence across all regions of the substrate. It iscontemplated, however, that in some embodiments spatially-directedsynthesis can be used to prepare a substrate having the firstoligonucleotide attached in selected regions of the substrate. Forexample, in some embodiments, a spatial array could be prepared havingmore than one first oligonucleotide segment sequence in differentlocations of the substrate.

Accordingly, as shown in FIGS. 34A-B that in at least one embodiment thecommon first oligonucleotide segment can be in situ synthesized directlyon the substrate however using light-directed synthesis with photolabilegroups, rather than standard protective groups, such as dimethoxytritylgroups. The synthesized first oligonucleotide segments would be preparedusing light-directed deprotection but would not be cleaved from thesubstrate or deprotected as shown in FIG. 34A. Following thislight-directed in situ synthesis of the first oligonucleotide segmentwith protective groups intact, light-directed spatial synthesis of thesecond oligonucleotide segment to the 3′-ends of the first segments canbe carried out as shown in FIG. 34B.

Generally, during standard automated oligonucleotide synthesis,following cleavage of the oligonucleotide from the synthesis solidsupport, the protecting groups on the phosphates and bases are removed(or deprotected) by treatment in concentrated aqueous ammonia with heat.Accordingly, it is also contemplated in at least one embodiment that afirst oligonucleotide is synthesized ex situ using standard automatedsynthesis, including cleavage and deprotection of the phosphodiestersand/or exocyclic amine moieties. As illustrated by the schematic of FIG.35A, the cleaved and deprotected first oligonucleotide segmentssynthesized ex situ can be attached to the substrate surface through alinkage to the 5′-end as described elsewhere herein. Then, as depictedby FIG. 35B, the phosphodiester and/or exocyclic amine moieties can beprotected in situ on the substrate. Following this in situ protectionstep, light-directed spatial synthesis of the second oligonucleotidesegment to the 3′-ends of the first segments can be carried out as shownin FIG. 35C.

In another embodiment illustrated by the schematic of FIG. 36A, ex situsynthesized first oligonucleotide segments can be cleaved from theirsynthesis support, deprotected, and then attached to a substrate surfacethrough a linkage to the 5′-end as described elsewhere herein. Then, asdepicted by FIG. 36B, a complementary oligonucleotide can be hybridizedto the common first oligonucleotide segments located across thesubstrate. The formation of a double-stranded structure by hybridizationcan effectively protect the phosphodiester and/or exocyclic aminemoieties of the first oligonucleotide segment on the substrate.Following this in situ protection-by-hybridization step, light-directedspatial synthesis of the second oligonucleotide segment to the 3′-endsof the first segments can be carried out as shown in FIG. 36C.

In at least one embodiment, the spatial array compositions preparedusing the methods of the present disclosure can be useful for spatialtranscriptomics applications as described in e.g., US2014/0066318A1.Briefly, in spatial transcriptomics a complex multi-cellular sample,such as a tissue sample, is immobilized on a spatial array ofoligonucleotides. Typically, the oligonucleotide sequences are attachedto the array substrate through their 5′-end and include an mRNA capturesequence at their 3′-end. Each of the oligonucleotides also includes5′-adjacent to the capture sequence, a random primer sequence of atleast 16-mer that allows for amplification of any captured sequences.The detection process results in amplification of the captured mRNAsequence as well as the barcode sequence. Sequencing of the ampliconsthereby indicates not only the identity of the transcribed gene but alsothe location in the multi-cellular sample where the gene wastranscribed.

Furthermore, in order to survey the genes undergoing transcription in acomplex multi-cellular sample, the spatial array should include anextremely high density of different oligonucleotides specificallylocated on the substrate. For example, it is contemplated that spatialarrays useful in various applications, such as spatial transcriptomics,can include 1.5 million, 10 million, 100 million, and even up to 1.0billion different oligonucleotides, each of which has a differentbarcode sequence and is located in a different region on a substrate,wherein the regions are less than 5 µm², even as small as 1.0 µm².

As noted elsewhere herein, each oligonucleotide of a spatial arrayshould comprise a different barcode sequence that correlates with itslocation. In some embodiments, it is contemplated that additionaloligonucleotides may be present on the same substrate as the spatialarray, that comprise a sequence identical to a barcode sequence of thespatial array. Such additional oligonucleotides may arise due to errorsin synthesis or can be prepared intentionally on the substrate (e.g.,for calibration or QC purposes). Generally, these sequences located in adifferent location on the same substrate are not considered part of thespatial array and are kept to a level and/or location that avoidsinterference with the intended function of the spatial array.

As illustrated by the particular exemplary application of spatialtranscriptomics, the ability to accurately correlate the barcodesequence of each oligonucleotide on a spatial array with a location (orregion) on the substrate is critical to the utility of the spatialarray. Accordingly, the preparation of spatial arrays requires methodsfor immobilizing up to a billion different oligonucleotides, each with aspecific barcode sequence, in a specific identifiable region on asubstrate.

Disclosed herein is a method of preparing a spatial array comprising (a)providing a solid support; (b) immobilizing a plurality of sets ofprimers on the solid support, wherein a first set of the plurality ofsets of primers comprises a first primer and a second primer, whereinthe first primer and the second primer are at a 1:1 ratio; and whereinthe first primer and the second primer are less than 10 µm apart; (c)providing a template oligonucleotide comprising a reverse complement ofa spatial barcode domain, a reverse complement of a capture domain, afirst hybridization domain capable of hybridizing to the first primer,and a second hybridization domain, wherein a reverse complement of thesecond hybridization domain is capable of hybridizing to the secondprimer; (d) hybridizing the template oligonucleotide to the firstprimer; (e) extending the first primer using the templateoligonucleotide as a template to generate a capture probe comprising thefirst primer, the spatial barcode, and the capture domain, which captureprobe is immobilized on the solid support; and (f) releasing thetemplate oligonucleotide from the spatial array. In some aspects, themethod further comprises amplifying the capture probe to create acluster of one or more capture probes. In some aspects, amplifyingcomprises: (a) hybridizing the capture probe to the second primer; (b)extending the second primer using the capture probe as a template togenerate a copy of the template oligonucleotide that is immobilized onthe solid support; (c) hybridizing the copy of the templateoligonucleotide to a first primer of a second set of the plurality ofprimers; and (d) extending the first primer of the second set of theplurality of primers using the copy of template oligonucleotide as atemplate to generate a copy of the capture probe that is immobilized onthe solid support, wherein the cluster of capture probes comprises thecapture probe and the copy of the capture probe.

In some aspects, the template oligonucleotide comprises a reversecomplement of a unique molecular identifier (UMI), and wherein thegenerated capture probe comprises the UMI.

In some aspects, the first primer is affixed to the solid support at a5′ end of the first primer.

In some aspects, the first primer comprises a sequence that iscomplementary to the template oligonucleotide.

In some aspects, the first primer comprises poly (A) domain.

In some aspects, the first primer comprises cleavage domain. In someaspects, the second primer is affixed to the solid support at a 5′ endof the second primer. In some aspects, the second primer comprises acleavage domain. In some aspects, the copy of the templateoligonucleotide is released by cleaving the cleavage domain after thecopy of the capture probe is generated.

In some aspects, the template oligonucleotide is attached to a bead. Insome aspects, the bead is a gel bead. In some aspects, the bead is 2microns or less in diameter. In some aspects, the bead is 1 micron orless in diameter. In some aspects, the bead is removed prior to the stepof extending the first primer. In some aspects, the bead is removedafter the step of extending the first primer.

In some aspects, the identity of the generated capture probe isdetermined. In some aspects, the identity of the generated capture probeis determined after the template oligonucleotide is released. In someaspects, the identity of the generated capture probe is determined insitu. In some aspects, the identity of the generated capture probe isdetermined by in situ sequencing. In some aspects, in situ sequencing isperformed via sequencing-by-synthesis (SBS), sequential fluorescencehybridization, sequencing by ligation, nucleic acid hybridization, orhigh-throughput digital sequencing techniques.

In some aspects, the capture domain comprises a sequence complementaryto an analyte of interest. In some aspects, the capture domain comprisesa poly(T) domain.

In some aspects, the solid support comprises a glass slide. In someaspects, the glass slide is a treated glass slide.

Also disclosed herein is a method for spatial analysis of a biologicalanalyte in a biological sample comprising: (a) preparing a spatial arrayby a method as provided herein; (b) decoding the one or more captureprobes by determining the identity of the spatial barcode of the one ormore capture probes, thereby associating the spatial barcode of the oneor more capture probe with a location on the spatial array; (c)contacting the biological sample to the spatial array under conditionswherein the biological analyte binds the cluster; (d) extending the oneor more capture probes via a polymerization reaction using thebiological analyte as a template to generate an extended one or morecapture probes comprising the one or more capture probes and the reversecomplement of the biological analyte; (e) determining the sequence ofthe extended one or more capture probes, and associating the spatialbarcode of the one or more capture probes with the captured biologicalanalyte; thereby determining spatial location of the biological analyte.

Also disclosed herein is a spatial array produced by any one of themethods as provided herein.

Further, disclosed herein is a spatial array comprising (a) a solidsupport; and (b) a plurality of sets of primers immobilized on the solidsupport, wherein a first set of the plurality of sets of primerscomprises a first primer and a second primer, wherein the first primerand the second primer are at a 1:1 ratio; wherein the first primer andthe second primer in each set are less than 10 µm apart; and wherein thefirst primer comprises a sequence that is complementary to a captureprobe. In some aspects, the spatial array further comprises a templateoligonucleotide comprising a reverse complement of a unique molecularidentifier (UMI), and wherein the capture probe comprises the UMI.

In some aspects, the first primer is affixed to the solid support at a5′ end of the first primer. In some aspects, the first primer comprisesa sequence that is complementary to the template oligonucleotide. Insome aspects, the first primer comprises poly (A) domain.

In some aspects, the first primer comprises cleavage domain. In someaspects, the second primer is affixed to the solid support at a 5′ endof the second primer. In some aspects, the second primer comprises acleavage domain.

In some aspects, the template oligonucleotide is attached to a bead. Insome aspects, the bead is a gel bead. In some aspects, the bead is 2microns or less in diameter. In some aspects, the bead is 1 micron orless in diameter.

In some aspects, the solid support comprises a glass slide. In someaspects, the glass slide is a treated glass slide.

Provided herein are methods of preparing a spatial array. A “spatialarray” as disclosed herein is an array that allows resolution equal toor less than 10 µm, allowing single-cell resolution. A spatial arraycomprises plurality of sets of primers on the solid support. The sets ofprimers comprises a sets of a first primer and a second primer at a 1:1ratio. The second primer has a sequence that is complementary to thefirst primer or to a template oligonucleotide.

The methods of preparing a spatial array include providing a solidsupport, immobilizing a plurality of sets of primers on the solidsupport, providing a template oligonucleotide having a reversecomplement of a spatial barcode domain, a reverse complement of acapture domain, a first hybridization domain capable of hybridizing tothe first primer, and a second hybridization domain, wherein a reversecomplement of the second hybridization domain is capable of hybridizingto the second primer; hybridizing the template oligonucleotide to thefirst primer; extending the first primer using the templateoligonucleotide as a template to generate a capture probe comprising thefirst primer, the spatial barcode, and the capture domain, which captureprobe is immobilized on the solid support; and releasing the templateoligonucleotide from the spatial array.

The spatial array comprises a plurality of sets of primers affixed tothe solid support. The plurality of sets of primers includes a firstprimer and a second primer that are at a 1:1 ratio and that are lessthan 10 µm apart. The first primer is affixed to the solid support atthe 5′ end of the first primer. The second primer is affixed to thesolid support at the 5′ end of the second primer.

The first primer and/or the second primer can be a nucleotide. In someaspects, the first primer is single stranded DNA. In some aspects, thefirst primer is RNA. In some aspects, the second primer is singlestranded DNA. In some aspects, the second primer is RNA.

The first primer on the solid support comprises a sequence that iscomplementary to the template oligonucleotide and allows the templateoligonucleotide to bind to the first primer. The first primer can alsocomprise a poly (A) or poly (T) domain that is complementary to apoly(T) or poly(A) domain of the second primer. The first primer canalso comprise a cleavage domain. The second primer can also comprise acleavage domain.

The first primer and the second primer are used to amplify a templateoligonucleotide to create a cluster of one or more capture probes. Insome aspects, the template oligonucleotide includes a reverse complementof a unique molecular identifier (UMI). Further, the templateoligonucleotide is attached to a bead. In some aspects, the bead is agel bead. In some aspects, the bead is less than 10 µm in diameter. Insome aspects, the bead is less than 5 µm in diameter. In some aspects,the bead is 2 µm in diameter. In some aspects, the bead is less than 2µm in diameter. In some aspects, the bead is 1 µm in diameter. In someaspects, the bead is less than 1 µm in diameter.

In some aspects, after binding of the template oligonucleotide to thefirst primer, the sequence of the primer is extended. In some aspects,the layer of beads that is attached to the oligos is removed afterextension of the primer. In some aspects, the layer of beads that isattached to the oligos is removed before extension of the primer.

After initial binding of the template oligonucleotide and the primers, acapture probe is generated. In some aspects, the capture probe isgenerated using cluster expansion.

In some aspects, the capture probe comprises a UMI. In some aspects, thecapture probe includes sequences for first primer, the spatial barcode,and the capture domain. In some aspects, the capture probe isimmobilized on the solid support. In some aspects, the capture domaincomprises a sequence complementary to an analyte of interest. In someaspects, the capture domain comprises a poly(T) domain.

After a copy of the capture probe is generated, the copy of the templateoligonucleotide is released by cleaving the cleavage domain after thecopy of the capture probe is generated.

After cluster amplification, the identity of the generated capture probeis determined. In some aspects, the identity of the generated captureprobe is determined after the template oligonucleotide is released. Insome aspects, the identity of the generated capture probe is determinedin situ. In some aspects, the identity of the generated capture probe isdetermined by in situ sequencing. In situ sequencing is performed viasequencing-by-synthesis (SBS), sequential fluorescence hybridization,sequencing by ligation, nucleic acid hybridization, or high-throughputdigital sequencing techniques.

After the probe is clonally expanded, spatial analysis of a biologicalanalyte in a biological sample can be performed. In some aspects, methodof performing spatial analysis of a biological analyte in a biologicalsample include preparing the spatial array as discussed above. In someaspects, the methods further include decoding the one or more captureprobes by determining the identity of the spatial barcode of the one ormore capture probes, thereby associating the spatial barcode of the oneor more capture probe with a location on the spatial array; contactingthe biological sample to the spatial array under conditions wherein thebiological analyte binds the cluster; extending the one or more captureprobes via a polymerization reaction using the biological analyte as atemplate to generate an extended one or more capture probes comprisingthe one or more capture probes and the reverse complement of thebiological analyte; and determining the sequence of the extended one ormore capture probes, and associating the spatial barcode of the one ormore capture probes with the captured biological analyte therebydetermining spatial location of the biological analyte.

EXAMPLE 1 - Method for Building an Array for Spatial Synthesis

In a non-limiting example, capture probes can be generated using aplurality of oligonucleotides for spatially barcoding analytes on anarray (FIG. 24 ). The oligonucleotides “A” include a constant sequence(A-i), and a spatial barcode sequence (A-ii), and optionally a secondconstant sequence (A-iii). A primer complementary to a portion of theoligonucleotide is hybridized with and extended using theoligonucleotide as a template to generate a first oligonucleotide. Thefirst oligonucleotide is further extended to create a 3′ end overhang,where an enzyme (e.g., template independent polymerase) is used tointroduce additional polynucleotides at the end of the firstoligonucleotide. A splint oligonucleotide “C” can be provided thathybridizes the 3′ end (e.g., overhang) of the first oligonucleotide. Asecond oligonucleotide is ligated to the 3′ end of the firstoligonucleotide to generate a capture probe.

As shown in FIG. 24 the second oligonucleotide “D” contains severaldomains. D-i contains a sequence complementary to a portion of thesplint oligonucleotide, D-ii is a degenerate sequence that can serve asa unique molecular identifier, and D-iii is a capture domain that can beused to capture an analyte in a biological sample. D-iii optionallycontains a poly-T sequence. Furthermore, D-iii can also be used toligate to the analyte in a biological sample. The capture probes can beused to spatially locate analytes according to the methods describedherein.

EXAMPLE 2 - Method for Spatial Analysis of a Biological Sample

In a non-limiting example, the array generated as described in Example 1is used for spatial analysis of a biological sample. The capture domain(D-iii) is used to hybridize with the poly-A tail of an mRNA moleculeSEQ ID NO: 8 (FIG. 25 ). Reverse transcription is carried out using areverse transcriptase enzyme to generate cDNA complementary to thecaptured mRNA. The sequence and location of the captured mRNA aredetermined by sequencing the capture probe that contains the spatialbarcode as well as the complementary cDNA.

EXAMPLE 3 - Methods for Preparing High-Density Spatially Barcoded Arrays

An array including one or more pluralities of first oligonucleotides iscontacted with a hydrogel including one or more pluralities of secondoligonucleotides. A first oligonucleotide can have a capture domain, asecond oligonucleotide can have a capture domain, or both first andsecond oligonucleotides can have a capture domain. Members of the one ormore pluralities of the first oligonucleotides are attached to membersof the one or more pluralities of second oligonucleotides to form one ormore pluralities of coupled capture probes (e.g., a combinatorial oligoarray is built). The size of the hydrogel containing the one or morepluralities of coupled capture probes is then reduced by removing water,which forms a shrunken hydrogel array containing the one or morepluralities of coupled capture probes (FIG. 26 ). The shrunken hydrogelarray can be contacted with a biological sample under conditionssufficient to allow analytes present in the biological sample tointeract with the coupled capture probes of the shrunken hydrogel array.

EXAMPLE 4 - Method for Building an Array Using a Two-Step BarcodeConstruction Process and a Protective Group

In a non-limiting example, a two-step spatial barcode constructionprocess is used to build an array with high complexity. An array thatincludes a plurality of features immobilized on a substrate is provided,where each feature includes a plurality of first oligonucleotides, andeach first oligonucleotide includes a first spatial barcode (FIG. 27 ).The first spatial barcode is associated with a particular feature, suchthat different features of the array are associated with differentspatial barcodes. The plurality of first oligonucleotides are attachedto a plurality of features or the substrate (e.g., direct attachment) atone end, and include a photocleavable protective group at the other end.A subset of the first oligonucleotides are exposed to a light source andthe protective group on the selected first oligonucleotides are removed,leaving the first oligonucleotides 3′ end available for attaching asecond oligonucleotide. Next, a second oligonucleotide is ligated to thefirst oligonucleotide that have had the protective groups removed, wherethe second oligonucleotide includes a second spatial barcode, a uniquemolecular identifier sequence, and a capture domain (e.g., a poly-Tsequence). In some cases, the second oligonucleotide also contains aprotective group at the 3′ end. The first oligonucleotide and the secondoligonucleotide can be ligated using a ligase (e.g., App ligase).Alternatively, the first oligonucleotide and the second oligonucleotideare ligated through a chemical step (e.g., similar to oligonucleotidesynthesis). Capture probes are thereby generated that contain the firstspatial barcode and the second spatial barcode. Optionally, theprotective group at the 3′ end of the capture probes may be removed togenerate a free 3′ —OH end.

EXAMPLE 5 - Method for Building an Array by Building Separate Sub-Arrays

In a non-limiting example, a two-step spatial barcode constructionprocess is used to build an array with high complexity. An array thatincludes a plurality of sub-arrays is provided, with each sub-arraycomprising a plurality of features immobilized on a substrate, whereeach feature includes a plurality of first oligonucleotides, and eachfirst oligonucleotide includes a first spatial barcode (BC_(A)). Thesub-array can include features wherein each feature is associated with afirst spatial barcode unique to the feature of origin on the sub-array.Therefore, any one particular feature of the sub-array associated with afirst spatial barcode, can be represented multiple times if thesub-array or subtemplate is printed at multiple locations (see FIGS. 29and 30 ). A plurality of second oligonucleotides are delivered to afirst sub-array, where each second oligonucleotide includes a secondspatial barcode (BC_(B)), a unique molecular identifier sequence, and acapture domain (e.g. a poly-T sequence). In some cases, the secondoligonucleotides also contain a protective group at the 3′ end. Thesecond oligonucleotides are ligated to the first oligonucleotides on thefirst subarray using a ligase, thereby generating capture probes thatinclude the first spatial barcode and the second spatial barcode.Alternatively, the first oligonucleotides and the secondoligonucleotides are ligated through a chemical step (e.g. similar tooligonucleotide synthesis). Optionally, the protective group at the 3′end of the capture probes may be removed to generate a 3′ —OH end.

Next, a second plurality of third oligonucleotides may be delivered to asecond subarray, where each third oligonucleotide includes a thirdspatial barcode, a unique molecular identifier sequence, and a secondcapture domain (e.g., a poly-T sequence). In some cases, the thirdoligonucleotides also contain a protective group at the 3′ end. Thethird oligonucleotides are ligated to a fourth oligonucleotidecontaining the first spatial barcode on the first sub-array using aligase, thereby generating capture probes that include the first spatialbarcode, the third spatial barcode, and the second capture domain.Optionally, the protective group at the 3′ end of the capture probes canbe removed to generate a 3′ - OH end.

EXAMPLE 6- Method for Preparing a Slide That Contains a High DensityArray

In a non-limiting example, the method of building a high density arrayas described in Examples 4 and 5 involves building the array on asubstrate (e.g., a slide). A slide is provided that includes a pluralityof identical sub-arrays, where each sub-array contains a plurality offirst oligonucleotides immobilized on the slide, and each firstoligonucleotide includes a first spatial barcode (BC_(A)). In somecases, there are no gaps between the sub-arrays.

In one example, the plurality of first oligonucleotides are attached tothe plurality of features or the substrate (e.g., direct attachment) atone end, and include a photocleavable protective group at the other end.A first sub-array is exposed to light while the remaining sub-arrays aremasked from the light. The protective group on the firstoligonucleotides on the first sub-array are thereby removed, leaving thefirst oligonucleotides available for attaching additionalpolynucleotides. Second oligonucleotides that contain a second spatialbarcode (BC_(B)) are attached to first oligonucleotides on the firstsub-array that have had the protective group removed to generate captureprobes that include both the first spatial barcode and second spatialbarcode. The second oligonucleotides further include a unique molecularidentifier and a capture domain (e.g. a poly-T sequence), and optionallya protective group attached to the 3′ end. Next, a second sub-array isexposed to light while the remaining sub-arrays are masked from thelight, and the above steps are repeated with third oligonucleotides thatcontain a third barcode (FIG. 29 ). Upon iteratively generating captureprobes in all sub-arrays on the slide, the 3′ protective group on thecapture probes are removed.

In one example, one or more physical barriers are used to isolate afirst subarray and second oligonucleotides that contain a second spatialbarcode are delivered to the isolated first sub-array using amicrofluidic device to generate capture probes that include both thefirst spatial barcode and the second spatial barcode. A second sub-arrayis then isolated and third oligonucleotides that contain a third spatialbarcode are delivered to the second sub-array to generate capture probesthat include both the first spatial barcode and the third spatialbarcode. Upon iteratively generating capture probes in all sub-arrays,the protective group at the 3′ end of the capture probes may be removedto generate a 3′ —OH end.

Fluidic isolation chambers can be used to isolate sub-arrays on a slide,such that oligonucleotides containing different barcodes can be appliedto different sub-arrays, as shown in FIG. 30 . The fluidic isolationchambers contain chamber walls that may further include a tapered endthat is highly hydrophobic and is designed to minimize footprint andallow for array continuity.

EXAMPLE 7 - Enrichment of a Spatial Barcode

A sequencing library structure is shown in FIG. 38 . A capture probeincluding an analyte, at least one spatial barcode, a region upstream ofthe at least one spatial barcode, and a region downstream of the analyteis enriched by the following method. A PCR reaction is initialized usinga primer with complementarity to the spatial barcode and another primerwith complementarity to a region (e.g., constant region 2) downstream ofthe analyte. The region may be a sequencing index or handle which has aknown and/or ubiquitous sequence such that the primer does not enrichcertain sequences over other sequences. A second round of PCR is thenrun by introducing another primer with complementarity to the regionupstream of the spatial barcode (e.g., constant region 1). This regionmay be a sequencing index or handle which has a known and/or ubiquitoussequence such that this primer does not enrich certain sequences overother sequences (FIG. 38 ). This exemplary method allows for theenrichment of the desired spatial barcode and analyte sequence whileretaining DNA regions upstream and downstream of the spatial barcodethat may be useful for any downstream DNA processing.

EXAMPLE 8 - Hierarchical Enrichment

Spatial enrichment can be performed on different pluralities of spatialbarcodes, wherein the different spatial barcodes correspond to regions,sub regions, and sub-sub regions. FIG. 37 shows an example wherein oneregion includes nine sub-regions, in which each sub-region furtherincludes fifteen sub-sub regions. In this example, each of the 135sub-sub regions possesses a spatial barcode (e.g., sub-sub spatialbarcode) unique to that sub-sub region, specifically including thespatial barcode of one of the nine subregions (sub spatial barcode), andthe spatial barcode of the region (first common sub spatial barcode).

An enrichment protocol can be performed using primers directed to atleast one, or more than one, spatial barcode (e.g., region spatialbarcode, sub spatial barcode, sub-sub spatial barcode). For example, alibrary can be created from the analytes captured by the array. Thelibrary can contain constant regions and spatial barcode regions.Primers can be employed to hybridize to the regional spatial barcode andamplify members of the library that contain the region specific spatialbarcode. Primers can be employed to hybridize to regional andsub-regional spatial barcodes and amplify members of the library thatcontain region specific and subregion specific spatial barcodes.Enriching multiple levels (e.g., region, subregion, sub-sub region) ofspatial barcodes can provide sequencing regions of interest at a lowercost.

EXAMPLE 9 - Spatial Enrichment

Capture probes with individual spatial barcodes will be linked to beadsand the beads are attached to create a high density bead array. Thecapture probes containing the spatial barcodes will be printed in 16identical arrays on a slide, wherein each array contains a plurality oftarget barcode sequences in a pre-defined pattern. Fresh, non-fixedmouse brain tissue will be trimmed and frozen in -40° C. cold isopentaneand subsequently will be mounted for sectioning with a cryostat at 10µm. A slice of tissue will be applied onto each capture probe array.

The SuperScript™ III One-step RT-PCR System with Platinum®Taq DNAPolymerase from Invitrogen can be used when performing the cDNAsynthesis. For each reaction 25 µl 2× reaction mix (SuperScript™ IIIOne-step RT-PCR System with Platinum®Taq DNA Polymerase, Invitrogen),22.5 µl H2O and 0.5 µl 100×BSA can be mixed and heated to 50° C.SuperScript III/Platinum Taq enzyme mix can be added to the reactionmix, 2 µl per reaction, and 50 µl of the 10 reaction mix can be added toeach well on the chip. The chip can be incubated at 50° C. for 30 min.

Prior to tissue application, fluorescent marker probes will behybridized to spot or beads comprising marker oligonucleotides printedon the capture probe array. The fluorescent marker probes can aid in theorientation of the resulting image after tissue visualization, making itpossible to combine the image with the resulting expression profiles forindividual capture probe spatial barcode sequences obtained aftersequencing. To hybridize fluorescent probes, a hybridization solutioncontaining 4×SSC and 0.1% SDS, 2 µM detection probe (P) will beincubated for 4 min at 50° C. Meanwhile, the high density bead array canbe attached to a ChipClip. The array can be subsequently incubated at50° C. for 30 min at 300 rpm shake with 50 µL of hybridization solutionper well.

Capture probes can be released using an enzyme mixture in TdT buffer orwith heated TdT buffer. For example, 38 µl of cleavage mixture can beplaced in a clean 0.2 ml PCR tube. The mixture can contain: 1 × TdTbuffer (20 mM Tris-acetate (pH 7.9), 50 mM Potassium Acetate and 10 mMMagnesium Acetate), 0.1 µg/µl BSA; 0.1 U/µl USER Enzyme; released cDNA;and released surface probes. To the PCR tube, 0.5 µl RNase H (5 U/µl,final concentration of 0.06 U/µl), 1 µl TdT (20 U/µl, finalconcentration of 0.5 U/µl), and 0.5 µl dATPs (100 mM, finalconcentration of 1.25 mM), can be added. For dA tailing, the tube can beincubated in a thermocycler at 37° C. for 15 min followed by aninactivation of TdT at 70° C. for 10 min. After dA tailing, a PCR mastermix can be prepared. The mix can contain: 1 × Faststart HiFi PCR Buffer(pH 8.3) with 1.8 mM MgCl2; 0.2 mM of each dNTP; 0.2 µM of each primer,A (complementary to the amplification domain of the capture probe) andB_(dT)24 (complementary to the poly-A tail to be added to the 3′ end ofthe first cDNA strand); and 0.1 U/µl Faststart HiFi DNA polymerase. 23µl of PCR Master mix can be placed into nine clean 0.2 ml PCR tubes. 2µl of dA tailing mixture can be added to eight of the tubes, while 2 µlwater (RNase/DNase free) can be added to the last tube (negativecontrol). PCR amplification can be carried out with the followingprogram: Hot start at 95° C. for 2 minutes, second strand synthesis at50° C. for 2 minutes and 72° C. for 3 5 minutes, amplification with 30PCR cycles at 95° C. for 30 seconds, 65° C. for 1 minutes, 72° C. for 3minutes, and a final extension at 72° C. for 10 minutes.

A dsDNA library for Illumina® sequencing using sample indexing can becarried out according to manufacturer’s instructions. Sequencing can becarried out on a HiSeq2000 platform. The sequencing data can beconverted from FASTQ format to FASTA format using the FastX toolkitFASTQ-to-FASTA converter. The sequencing reads can be aligned to thecapture probe positional domain (tag) sequences using Blastn and thereads with hits better than 1e-6 to one of tag sequences were sorted outto individual files for each tag sequence respectively. The file of tagsequence reads can then be aligned using Blastn to the mousetranscriptome, and hits collected.

The expression profiles for individual capture probe spatial barcodesequences can be combined with the spatial information obtained from thetissue sections through staining. The transcriptomic data from thecellular compartments of the tissue section can be analyzed in adirectly comparative fashion, with the availability to distinguishdistinct expression features for different cellular subtypes in a givenstructural context.

EXAMPLE 10 - Generating Gene Specific Capture Probes

Capture probes containing various functional sequences and gene specificor general (e.g., poly(T)) capture domains were generated with aligation strategy. FIG. 41 shows an immobilized oligonucleotide,acceptor oligonucleotide, on a substrate, from 3′ to 5′ containing anamino modifier C6 at its 3′ end, followed by 12 uracils that canfunction as a cleavage domain, a sequencing handle (e.g., R1), a 16 basepair spatial barcode, a 12 base pair UMI, and a ligation handle (SEQ IDNO: 9). A splint oligonucleotide (SEQ ID NO: 10) with a sequencecomplementary to the ligation handle and an additional sequence (e.g.,poly(A) or gene specific sequence) facilitates the ligation ofoligonucleotide and the capture domain (SEQ ID NO: 11). Ligation of bothpoly(T) (e.g., mRNA) and a specific mRNA (e.g., Penk) capture domains tothe immobilized oligonucleotide was completed using a mixture containing30 µM Poly dT or capture sequence (e.g., Penk), 30 µM splintoligonucleotide, and 100 units of ligase. The mixture was placed on topof the slide for 60 minutes then removed before washing and addingtissue. All tissues were permeabilized without pre-permeabilization fora duration of 3, 6, or 9 minutes (FIG. 43 ). The data show Poly dT wassuccessfully ligated to the immobilized oligonucleotide and capable ofcapturing mRNA in a biased manner. Additionally, a capture domain forthe Penk mRNA was successfully ligated and capable of capturing PenkmRNA. As shown in the table below, ligating a Poly dT capture domain didnot affect sequencing efficiency. The reads mapped to the transcriptomefor gene specific data are relatively high for a single gene capture(e.g., Penk), but the ligation strategy and subsequent gene specificmRNA capture is possible.

Name Reads Mapped Confidently to Transcriptoma Reads Mapped Confidentlyto Genome Fraction reads with primer or homopolymer sequence Fractionreads with any polyA sequence Fraction ribosomal protein UMI countsFraction mitochondrial UMI counts Ligated PolyT (N=6) 81.42% 90.94%13.98% 4.30% 9.94% 25.80% No Ligate Average (N=8) 83.12% 92.67% 13.37%2.52% 10.38% 26.15% Pank Average (N=6) 13.33% 53.07% 10.78% 0.28% 1.18%2.78%

EXAMPLE 11 - Ligation of Capture Domains to Immobilized Oligonucleotides

The following ligation protocol and reagents provide for the ligation ofcapture domains to existing arrays. A portion of a splintoligonucleotide is complementary to the ligation handle of an acceptoroligonucleotide, acceptor oligonucleotide 2, on the surface of thearray. Another portion of the splint oligonucleotide is complementary toa second ligation handle sequence present in an oligonucleotide thatalso includes the capture domain. The splint oligonucleotide facilitatesthe ligation of the capture domain (e.g., via hybridization to thesecond ligation handle) to the immobilized oligonucleotide (e.g.,ligation handle of the immobilized oligonucleotide).

Ligation Protocol

Equal amounts of a splint oligonucleotide and a capture domain areincubated at room temperature overnight. Alternatively, equal amounts ofa splint oligonucleotide and a ligation probe are mixed and warmed up to98° C. for 2 minutes, and then cooled down to 4° C. at a rate of 0. 1°C./second.

Next, 100 µl probe and ligase mix are added to each well, and incubatedat 22° C. for 60 minutes. After 60 minutes, 100 µl of volume is removedand discard. 100 µl of 2X SSC buffer that has been heated to 50° C. isadded to each well and incubated for 2 minutes. After 2 minutes, 100 µlof the 2X SSC buffer is removed from the well and discard. This processis repeated 2 times for a total of 3 washes in 2X SSC buffer at 50° C.

After the 3 washes in 2X SSC buffer, 100 µl of room temperature 0.2X SSCbuffer is added to each well and incubated for 1 minute. 100 µl of 0.2XSSC buffer is then removed, followed by addition of 100 µl of 0.1X SSCbuffer.

The slide can be stored at 4° C. until ready for fluorescent probehybridization and imaging.

Ligation reaction reagent mix Stock Concentration Desired ConcentrationVolume (µl) Desired final volume 100 H₂O 55 ATP 100 2.5 2.5 5X LigationBuffer 5X 1X 20 Ligase 1000 100 10 PolyT + splint oligonucleotide 250 3012 Reagents in the Ligation reaction reagent mix table Item 10x PartNumber Origin Composition 5X Ligation Buffer 210007 10x Manufactured(bulk formulation) 35% Glycerol, 250 mM Tris-HCl pH 7.5, 50 mM MgCl₂Bulk, ATP 100 mM 210010 10x Manufactured (bulk formulation) 100 mM(powder disodium salt dissolved in Nuclease-Free Water). pH adjusted to6.8-7.2 using 10 M NaOH DTT-Free T4 Ligase 310280 Enzymatics (Qiagen)Buffer: 10 mM Tris-HCl, 50 mM KCl, 0.1 mM EDTA, 50% glycerol, pH 7.5

SEQUENCE LISTING

Synthetic PURAMATRIX polypeptide sequenceSEQ ID NO : 1 RADARADARADARADA

Synthetic EAK16 polypeptide sequenceSEQ ID NO: 2 A EAEAKAKAEAEAKAK

Synthetic KLD12 polypeptide sequenceSEQ ID NO: 3 K LDLKLDLKLDL

18s cDNA Probe 1 (P1)SEQ ID NO: 4 GAGGAATTCCCAGTAA GT

18s cDNA Probe 2 (P2)SEQ ID NO: 5 GAGATTGAGCAATAAC AG

18s cDNA Probe 3 (P3)SEQ ID NO: 6 GTAGTTCCGACCATAA AC

18s cDNA Probe 4 (P4)SEQ ID NO: 7 GGTGACTCTAGATAAC CT

Analyte poly(A) tail from a biological sampleSEQ ID NO: 8 AAAAAAAAAAANNNNNNNNNNNNNNNNNNNNNNNN

Acceptor OligonucleotideSEQ ID NO: 9UUUUUUUUUUUUCTACACGACTCTTCCGATCTACGTACGTACGTACGTNNNNNNNNNNNNGCGG CCCG

Splint oligonucleotideSEQ ID NO: 10 AAAAAAAAAACGGG CCGC

Capture domain poly(T) sequenceSEQ ID NO: 11 TTTTTTTTTTTTTTTTTTTTTTTTTTT

Acceptor Oligonucleotide 2SEQ ID NO: 12TTTTTCTACACGACGCTCTTCCGATCTACGTACGTACGTACGTNNNNNNNNNNNNCGCTAC G

What is claimed is:
 1. (canceled)
 2. A method of generating a spatialarray comprising: (a) immobilizing a first plurality of primers and asecond plurality of primers on a substrate, wherein: (i) a first primerof the first plurality of primers comprises one or more functionaldomains and a sequence complementary to a hybridization domain; and (ii)a second primer of the second plurality of primers comprises a sequencecomplementary to a capture domain; (b) providing a templateoligonucleotide comprising the hybridization domain capable ofhybridizing to the sequence complementary to the hybridization domain ofthe first primer, a complement of a spatial barcode, and a complement ofthe capture domain; (c) hybridizing the hybridization domain of thetemplate oligonucleotide to the sequence complementary to thehybridization domain of the first primer; (d) extending the first primerusing the template oligonucleotide as a template to generate a captureprobe comprising the first primer, the spatial barcode, and the capturedomain; and (e) releasing the template oligonucleotide from the captureprobe, thereby generating the spatial array.
 3. The method of claim 2,further comprising amplifying the capture probe to create a clonalcluster of capture probes.
 4. The method of claim 3, wherein theamplifying comprises: (a) hybridizing the capture domain of the captureprobe to the sequence complementary to the capture domain of the secondprimer; (b) extending the second primer using the capture probe as atemplate to generate a copy of the template oligonucleotide immobilizedon the substrate; (c) releasing the copy of the template oligonucleotidefrom the capture probe; (d) hybridizing the copy of the templateoligonucleotide to an unextended first primer; and (e) extending theunextended first primer using the copy of template oligonucleotide as atemplate to generate a second capture probe immobilized on thesubstrate, thereby generating the clonal cluster of capture probes. 5.The method of claim 4, further comprising extending the templateoligonucleotide using the first primer as a template.
 6. The method ofclaim 2, wherein the template oligonucleotide further comprises a uniquemolecular identifier (UMI) and one or more functional domains.
 7. Themethod of claim 6, wherein the one or more functional domains of thefirst primer and/or the template oligonucleotide comprise a sequencingspecific site or a primer binding site.
 8. The method of claim 6,wherein the UMI is disposed between the complement of the spatialbarcode and the complement of the capture domain.
 9. The method of claim2, wherein the first primer and the second primer are immobilized to thesubstrate at their 5′ ends.
 10. The method of claim 2, wherein the firstprimer and the second primer are at a ratio between 1:1.5 and 1.5:1. 11.The method of claim 2, wherein the first primer and the second primerare less than 5 µm apart.
 12. The method of claim 2, wherein thesequence complementary to the capture domain of the second primercomprises a poly(A) sequence.
 13. The method of claim 4, wherein thesecond primer further comprises a cleavage domain.
 14. The method ofclaim 13, wherein releasing the copy of the template oligonucleotidecomprises cleaving the cleavage domain of the second primer anddenaturing the copy of the template oligonucleotide from the captureprobe.
 15. The method of claim 2, wherein the template oligonucleotideis attached to a bead.
 16. The method of claim 15, wherein the bead is agel bead.
 17. The method of claim 15, wherein the bead is 2 microns orless in diameter.
 18. The method of claim 17, wherein the bead is 1micron or less in diameter.
 19. The method of claim 15, wherein the beadis removed after the step of extending the first primer.
 20. The methodof claim 15, wherein the bead is removed between steps (c) and (d). 21.The method of claim 15, wherein the bead is comprised in a plurality ofbeads dispersed on the substrate such that a single layer of beads isdisposed on the substrate.
 22. The method of claim 2, wherein thecapture domain comprises a poly(T) sequence.
 23. The method of claim 22,wherein the poly(T) sequence hybridizes to an analyte.
 24. The method ofclaim 23, wherein the analyte is mRNA.
 25. The method of claim 24,further comprising extending the capture probe using the mRNA as atemplate to generate an extended capture probe.
 26. The method of claim2, wherein the substrate comprises a glass slide.
 27. The method ofclaim 26, wherein the glass slide is a treated glass slide.
 28. Themethod of claim 2, further comprising determining the sequence of thespatial barcode of the generated capture probe.
 29. The method of claim28, wherein determining the sequence of the spatial barcode of thegenerated capture probe is performed after the template oligonucleotideis released.
 30. The method of claim 29, wherein determining thesequence of the spatial barcode of the generated capture probe comprisessequencing.
 31. The method of claim 30, wherein the sequencing comprisessequencing-by-synthesis (SBS), sequencing-by-hybridization (SBH), orsequencing-by-ligation (SBL).